Compositions and methods for monitoring the phosphorylation of natural binding partners

ABSTRACT

This invention relates to methods and compositions for monitoring the interaction of binding partners as a function of the addition or subtraction of a phosphate group to or from one of the binding partners by a protein kinase or phosphatase.

FIELD OF THE INVENTION

[0001] The invention relates to monitoring of phosphorylation ordephosphorylation of a protein.

BACKGROUND OF THE INVENTION

[0002] The post-translational modification of proteins has been knownfor over 40 years and since then has become a ubiquitous feature ofprotein structure. The addition of biochemical groups to translatedpolypeptides has wide-ranging effects on protein stability, proteinsecondary/tertiary structure, enzyme activity and in more general termson the regulated homeostasis of cells. Such additions include, but arenot limited to, protein phosphorylation and dephosphorylation.

[0003] Phosphorylation is a well-studied example of a post-translationalmodification of proteins. There are many cases in which polypeptidesform higher order tertiary structures with like polypeptides(homo-oligomers) or with unalike polypeptides (hetero-oligomers). In thesimplest scenario, two identical polypeptides associate to form anactive homodimer. An example of this type of association is the naturalassociation of myosin II molecules in the assembly of myosin intofilaments.

[0004] The dimerization of myosin II monomers is the initial step inseeding myosin filaments. The initial dimerization is regulated byphosphorylation, the effect of which is to induce a conformationalchange in myosin II secondary structure resulting in the folded 10Smonomer subunit extending to a 6S molecule. This active molecule is ableto dimerize and subsequently to form filaments. The involvement ofphosphorylation of myosin II in this priming event is somewhatcontroversial. Although in higher eukaryotes the conformational changeis dependant on phosphorylation, in Ancanthoamoeba, a lower eukaryote,the post-translational addition of phosphate is not required to effectthe initial dimerization step. It is of note that the dimerizationdomains in myosin II of higher eukaryotes contain the sites forphosphorylation and it is probable that phosphorylation in this regionis responsible for enabling myosin II to dimerize and subsequently formfilaments. In Dictyostelium this situation is reversed in that thephosphorylation sites are outside the dimerization domain andphosphorylation at these sites is required to effect the disassembly ofmyosin filaments. In contrast to both these examples, Acanthoamoebamyosin II is phosphorylated in the dimerization domain but thismodification is not necessary to enable myosin II monomers to dimerizein this species.

[0005] By far the most frequent example of post-translationalmodification is the addition of phosphate to polypeptides by specificenzymes known as protein kinases. These enzymes have been identified asimportant regulators of the state of phosphorylation of target proteinsand have been implicated as major players in regulating cellularphysiology. For example, the cell-division-cycle of the eukaryotic cellis primarily regulated by the state of phosphorylation of specificproteins, the functional state of which is determined by whether or notthe protein is phosphorylated. This is determined by the relativeactivity of protein kinases which add phosphate and protein phosphataseswhich remove the phosphate moiety from these proteins. Clearlydysfunction of either the kinases or phosphatases may lead to a diseasedstate. This is best exemplified by the uncontrolled cellular divisionshown by tumor cells. The regulatory pathway is composed of a largenumber of genes that interact in vivo to regulate the phosphorylationcascade that ultimately determines if a cell is to divide or arrest celldivision.

[0006] Currently there are several approaches to analysing the state ofmodification of target proteins in vivo:

[0007] 1. In vivo incorporation of labeled (for example, radiolabeled)phosphate, which is added to target proteins. According to one commonprocedure, intracellular ATP pools are labeled with ³²PO₄, which issubsequently incorporated into protein. Analysis of modified proteins istypically performed by electrophoresis and autoradiography, withspecificity enhanced by immunoprecipitation of proteins of interestprior to electrophoresis.

[0008] 2. Back-labeling. The incorporation of a labeled phosphate (e.g.,³²P) into a protein in vitro to estimate the state of modification invivo.

[0009] 3. The use of cell membrane-permeable protein kinase inhibitors(e.g., Wortmannin, staurosporine) to block phosphorylation of targetproteins.

[0010] 4. Western blotting, of either 1- or 2-dimensional gels bearingtest protein samples, in which phosphorylation is detected usingantibodies specific for phosphorylated forms of target proteins.

[0011] 5. The exploitation of eukaryotic microbial systems to identifymutations in protein kinases and/or protein phosphatases.

[0012] These strategies have certain limitations. Monitoring states ofphosphorylation by pulse or steady-state labeling is merely adescriptive strategy to show which proteins are phosphorylated whensamples are separated by gel electrophoresis and visualized byautoradiography. This is unsatisfactory, due to the inability toidentify many of the proteins that are phosphorylated. A degree ofspecificity is afforded to this technique if it is combined withimmunoprecipitation; however, this is of course limited by theavailability of antibodies to target proteins. Moreover, onlyhighly-expressed proteins are readily detectable using this technique,which may fail to identify many low-abundance proteins, which arepotentially important regulators of cellular functions.

[0013] The use of kinase inhibitors to block activity is alsoproblematic. For example, very few kinase inhibitors have adequatespecificity to allow for the unequivocal correlation of a given kinasewith a specific kinase reaction. Indeed, many inhibitors have a broadinhibitory range. For example, staurosporine is a potent inhibitor ofphospholipid/Ca⁺² dependant kinases. Wortmannin is some what morespecific, being limited to the phosphatidylinositol-3 kinase family.This is clearly unsatisfactory because more than one biochemical pathwaymay be affected during treatment making the assignment of the effectsalmost impossible.

[0014] Monoclonal antibodies directed against phosphorylated epitopes,except in specific cases, exhibit a limitation of specificity comparableto that observed when in vivo labeling is undertaken. Immunologicalmethods can only detect phosphorylated proteins globally (e.g., ananti-phosphotyrosine antibody will detect all tyrosine-phosphorylatedproteins) and can only describe a steady state, rather provide areal-time assessment of protein:protein interactions. Such assays alsorequire considerable manpower for processing.

[0015] Finally, yeast (Saccharomyces cervisiae and Schizosaccharomycespombe) has been exploited as a model organism for the identification ofgene function using recessive mutations. It is through research on theeffects of these mutations that the functional specificities of manyprotein kinases have been elucidated. However, these molecular genetictechniques are not easily transferable to higher eukaryotes, which arediploid and therefore not as genetically tractable as these lowereukaryotes.

[0016] Recent research into the sites of protein phosphorylation hasrevealed a number of sequence specific motifs which, when phosphorylatedor dephosphorylated, promote interaction with selected target proteinsto either induce or inhibit activity of either the phosphorylatedpolypeptide or the target polypeptide.

[0017] For example, and not by way of limitation, many proteins involvedin intracellular signal transduction have been shown to contain a domaincomprising a sequence of approximately 100 amino acids; this sequence istermed the Src homology two (SH2) domain. SH2 domains bind targetpolypeptides that contain phosphorylated tyrosine. This binding isdependent on the primary amino acid sequence around the phosphotyrosinein the target protein and several peptide sequences which, whenphosphorylated, bind to an SH2 domain have been identified (see e.g.,Songyang et al., 1993, Cell, 72: 767-778). Non-limiting examples of suchsequences include FLPVPEYINQSV, a sequence found in human ECF receptor,and AVGNPEYLNTVQ, a sequence found in human EGF receptor, both of whichare autophosphorylated growth factor receptors which stimulate thebiochemical signaling pathways that control gene expression,cytoskeletal architecture and cell metabolism. Both of these sequencesinteract with SH2 domains found in the Sen-5 adapter protein.

[0018] The tumor suppressor protein p53, becomes activated by atranscription factor in response to DNA damage. A DNA-dependent proteinkinase (DNA-PK) that is activated in response to breaks in DNA isthought to be regulator of p53 activity (Woo et al., 1998, Nature, 394:700-704). The data described by Woo et al. indicate that thephosphorylation of p53 by DNA-PK serves a dual purpose insofar asphosphorylation promotes the binding of p53 to DNA and also prevents p53inactivation by MDM2. A p53-derived peptide sequence EPPLSOEAFADLWKK isidentified as the site of phosphorylation in p53 that (whenphosphorylated) prevents the interaction of p53 with MDM2.

[0019] An example of heterodimer association is described in patentapplication number WO92/00388. It describes an adenosine 3:5 cylicmonophosphate (cAMP) dependent protein kinase which is a four-subunitenzyme being composed of two catalytic polypeptides (C) and tworegulatory polypeptides (R). In nature the polypeptides associate in astoichiometry of R₂C₂. In the absence of cAMP the R and C subunitsassociate and the enzyme complex is inactive. In the presence of cAMPthe R subunit functions as a ligand for cAMP resulting in dissociationof the complex and the release of active protein kinase. The inventiondescribed in WO92/00388 exploits this association by addingfluorochromes to the R and C subunits.

[0020] The polypeptides are labeled (or ‘tagged’) with fluorophoreshaving different excitation/emission wavelengths. The excitation andemission of one such fluorophore effects a second excitation/emissionevent in the second fluorophore. By monitoring the fluorescence emissionof each fluorophore, which reflects the presence or absence offluorescence energy transfer between the two, it is possible to derivethe concentration of cAMP as a function of the level of associationbetween the R and C. Therefore, the natural affinity of the C subunitfor the R subunit has been exploited to monitor the concentration of aspecific metabolite, namely cAMP.

[0021] The prior art teaches that intact, fluorophore-labeled proteinscan function as reporter molecules for monitoring the formation ofmulti-subunit complexes from protein monomers; however, in each case,the technique relies on the natural ability of the protein monomers toassociate.

[0022] Tsien et al. (WO97/28261) teach that fluorescent proteins havingthe proper emission and excitation spectra that are brought intophysically close proximity with one another can exhibit fluorescenceresonance energy transfer (“FRET”). The invention of WO97/28261 takesadvantage of that discovery to provide tandem fluorescent proteinconstructs in which two fluorescent protein labels capable of exhibitingFRET are coupled through a linker to form a tandem construct. In theassays of Tsien et al., protease activity is monitored using FRET todetermine the distance between fluorophores controlled by a peptidelinker and subsequent hydrolysis thereof. Other applications rely on achange in the intrinsic fluorescence of the protein as in the kinaseassays of WO98/06737.

[0023] The present invention instead encompasses monitoring of theassociation of polypeptides, as described herein, which are labeled withfluorescent (protein and chemical) or other labels. FRET, a non-limitingexample of a detection method of use in the invention, indicates theproximity of two labeled polypeptide binding partners, which labeledpartners associate either in the presence or absence ofpost-translational addition/removal of a phosphate group to/from anatural binding domain present in at least one of the partners, but notinto the fluorophore, reflecting the phosphorylation state of one orboth of the binding partners and, consequently, the level of activity ofa protein kinase or phosphatase.

[0024] There is a need in the art for efficient means of monitoringand/or modulating post-translational protein phosphorylation and/ordephosphorylation. Further, there is a need to develop a techniquewhereby the addition/removal of a phosphate group can be monitoredcontinuously during real time to provide a dynamic assay system thatalso has the ability to resolve spatial information.

SUMMARY OF THE INVENTION

[0025] The invention provides natural binding domains, sequences andpolypeptides, as well as kits comprising these molecules and assays ofenzymatic function in which they are employed as reporter molecules. Asused herein in reference to a polypeptide component of assays of theinvention, the term “natural” refers both to the existence of such anamino acid sequence, whether contiguous or non-contiguous, in nature aswell as to the phosphorylation-dependent binding of that component to asecond polypeptide or binding partner, and does not relate to attributesof such a polypeptide other than such binding.

[0026] One aspect of the invention is an isolated natural binding domainand a binding partner therefor, wherein the isolated natural bindingdomain includes a site for post-translational phosphorylation and bindsthe binding partner in a manner dependent upon phosphorylation ordephosphorylation of the site.

[0027] The invention also provides a method for monitoring activity ofan enzyme comprising performing a detection step to detect binding of anisolated natural binding domain and a binding partner therefor as aresult of contacting one or both of the isolated natural binding domainand the binding partner with the enzyme, wherein the isolated naturalbinding domain includes a site for post-translational phosphorylationand binds the binding partner in a manner dependent upon phosphorylationof the site and wherein detection of binding of the isolated naturalbinding domain and the binding partner as a result of the contacting isindicative of enzyme activity.

[0028] An enzyme to be assayed according to the invention is a proteinkinase or a phosphatase.

[0029] The invention additionally encompasses a method for monitoringactivity of an enzyme comprising performing a detection step to detectdissociation of an isolated natural binding domain from a bindingpartner therefor as a result of contacting one or both of the isolatednatural binding domain and the binding partner with the enzyme, whereinthe isolated natural binding domain includes a site forpost-translational phosphorylation and binds the binding partner in amanner dependent upon phosphorylation of the site and wherein detectionof dissociation of the isolated natural binding domain from the bindingpartner as a result of the contacting is indicative of enzyme activity.

[0030] As used herein, the term “binding domain” in a three-dimensionalsense refers to the amino acid residues of a first polypeptide requiredfor phosphorylation-dependent binding between the first polypeptide andits binding partner. The amino acids of a “binding domain” may be eithercontiguous or non-contiguous and may form a binding pocket forphosphorylation-dependent binding. A domain must include at least 1amino acid, but may include 2 or more amino acids, preferably at least 4amino acids, which are contiguous or non-contiguous, but are necessaryfor phosphorylation-dependent binding to the binding partner. A bindingdomain will not include a natural full-length polypeptide, but willinclude a subset of the amino acids of a full-length polypeptide,wherein the subset may include a number of amino acids as high as onefewer than the length of a given natural full-length polypeptide.

[0031] A binding domain which is of use in the invention is a “naturalbinding domain” (i.e., a binding domain that exhibitsphosphorylation-dependent binding to a binding partner in nature). Anatural binding domain of use in the invention may be isolated or may bepresent in the context of a larger polypeptide molecule (i.e., one whichcomprises amino acids other than those of the natural binding domain),which molecule may be either naturally-occurring or recombinant and, inthe case of the latter, may comprise either natural or non-natural aminoacid sequences outside the binding domain.

[0032] As used herein with regard to phosphorylation or dephosphoylationof a polypeptide, the term “site” refers to an amino acid or amino acidsequence of a natural binding domain or a binding partner which isrecognized by (i.e., a signal for) a kinase or phosphatase for thepurpose of phosphorylation or dephosphorylation (i.e., addition orremoval of a phosphate moiety) of the polypeptide or a portion thereof.A “site” additionally refers to the single amino acid which isphosphorylated or dephosphorylated. It is contemplated that a sitecomprises a small number of amino acids, as few as one but typicallyfrom 2 to 10, less often up to 30 amino acids, and further that a sitecomprises fewer than the total number of amino acids present in thepolypeptide.

[0033] In an enzymatic assay of the invention, a “site”, forpost-translational phosphorylation or dephosphorylation may be presenton either or both of the isolated natural binding domain or the bindingpartner therefor. If such sites are present on both the isolated naturalbinding domain and its binding partner, binding between the naturalbinding domain and the binding partner, or between two natural bindingdomains, may be dependent upon the phosphorylation or dephosphorylationstate of either one or both sites. If a single polypeptide chaincomprises the natural binding domain and the binding partner (or twonatural binding domains), the state of phosphorylation ordephosphorylation of one or both sites will determine whether bindingoccurs.

[0034] A site suitable for addition or removal of a phosphate moiety ispresent within an isolated natural binding domain or binding partnerthereof of the invention at a position such that formation of a complexbetween the isolated natural binding domain and its binding partner isdependent upon the presence or absence of the phosphate moiety; andpreferably does not overlap with an amino acid which is part of afluorescent tag or other detectable label (including, but not limitedto, a radioactive label) or quencher.

[0035] Similarly, the amino acid that includes a phosphate moiety may bepositioned anywhere within the isolated natural binding domain such thatbinding of the isolated natural binding domain and its binding partneris dependent upon the presence or absence of the phosphate moiety.

[0036] As used herein, “phosphorylation” and “dephosphorylation” referto the addition or removal of a phosphate moiety to/from a polypeptide,respectively. As used herein, the term “post-translational modification”refers to the addition or removal of a phosphate moiety and does notrefer to other post-translational events which do not involve additionor removal of a phosphate moiety, and thus does not include simplecleavage of the reporter molecule polypeptide backbone by hydrolysis ofa peptide bond.

[0037] As used herein, the term “moiety” refers to apost-translationally added or removed phosphate (PO₄) group; the terms“moiety” and “group” are used interchangeably.

[0038] As used herein, the term “binding partner” refers to apolypeptide or fragment thereof (a peptide) that binds to a bindingdomain, sequence or polypeptide, as defined herein, in a manner which isdependent upon the state of phosphorylation of a site forphosphorylation or dephosphorylation which is, at a minimum, presentupon the binding domain, sequence or polypeptide; the binding partneritself may, optionally, comprise such a site and binding between thebinding domain, fragment or polypeptide with its corresponding bindingpartner may, optionally, depend upon modification of that site. Abinding partner does not necessarily have to contain a site forphosphorylation or dephosphorylation if such an site is not required tobe present on it for modification-dependent association between it and abinding domain, sequence or polypeptide. Binding partners of use in theinvention are those which are found in nature and exhibit naturalphosphorylation-dependent binding to a natural binding domain, sequenceor polypeptide of the invention as defined herein. In one embodiment ofthe invention, a binding partner is shorter (i.e., by at least oneN-terminal or C-terminal amino acid) than the natural full-lengthpolypeptide.

[0039] As used herein, the term “associates” or “binds” refers to anatural binding domain as described herein and its binding partner,having a binding constant sufficiently strong to allow detection ofbinding by FRET or other detection means, which are in physical contactwith each other and have a dissociation constant (Kd) of about 10 μM orlower. The contact region may include all or parts of the two molecules.Therefore, the terms “substantially dissociated” and “dissociated” or“substantially unbound” or “unbound” refer to the absence or loss ofcontact between such regions, such that the binding constant is reducedby an amount which produces a discernable change in a signal compared tothe bound state, including a total absence or loss of contact, such thatthe proteins are completely separated, as well as a partial absence orloss of contact, so that the body of the proteins are no longer in closeproximity to each other but may still be tethered together or otherwiseloosely attached, and thus have a dissociation constant greater than 10μM (Kd). In many cases, the Kd will be in the mM range. The terms“complex”, “dimer”, “multimer” and “oligomer” as used herein, refer tothe natural binding domain and its binding partner in the associated orbound state. More than one molecule of each of the two or more proteinsmay be present in a complex, dimer, multimer or oligomer according tothe methods of the invention.

[0040] As used herein in reference to a natural binding domain or otherpolypeptide, the term “isolated” refers to a molecule or population ofmolecules that is substantially pure (i.e., free of contaminatingmolecules of unlike amino acid sequence).

[0041] As used herein in reference to the purity of a molecule orpopulation thereof, the term “substantially” refers to that which is atleast 50%, preferably 60-75%, more preferably from 80-95% and, mostpreferably, from 98-100% pure.

[0042] “Naturally-occurring” as used herein, as applied to a polypeptideor polynucleotide, refers to the fact that the polypeptide orpolynucleotide can be found in nature. One such example is a polypeptideor polynucleotide sequence that is present in an organism (including avirus) that can be isolated form a source in nature.

[0043] The term “synthetic”, as used herein, is defined as any amino- ornucleic acid sequence which is produced via chemical synthesis.

[0044] In an assay of the invention, post-translational phosphorylationis reversible, such that repeating cycles of addition and removal of aphosphate moiety may be observed, although such cycles may not occur ina living cell found in nature.

[0045] An advantage of assays of the invention is that they may, ifdesired, be performed in “real time”. As used herein in reference tomonitoring, measurements or observations in assays of the invention, theterm “real time” refers to that which is performed contemporaneouslywith the monitored, measured or observed events and which yields aresult of the monitoring, measurement or observation to one who performsit simultaneously, or effectively so, with the occurrence of amonitored, measured or observed event. Thus, a “real time” assay ormeasurement contains not only the measured and quantitated result, suchas fluorescence, but expresses this in real time, that is, in hours,minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shortertimes exceed the instrumentation capability; further, resolution is alsolimited by the folding and binding kinetics of polypeptides.

[0046] As used herein, the term “binding sequence” refers to thatportion of a polypeptide comprising at least 1, preferably at least 2,more preferably at least 4, and up to 8, 10, 100 or even 1000 contiguous(i.e., covalently linked by peptide bonds) amino acid residues, that aresufficient for phosphorylation-dependent binding to a binding partner. Abinding sequence will not include a natural full-length polypeptide, butwill include a subset of the amino acids of a full-length polypeptide,wherein the subset may include a number of amino acids as high as onefewer than the length of a given natural full-length polypeptide.

[0047] As used herein in reference to those binding sequences that areof use in the invention, the term “natural binding sequence” refers to abinding sequence, as defined above, which consists of an amino acidsequence which is found in nature and which is naturally dependent uponthe phosphorylation state of a site for post-translationalphosphorylation found within it for binding to a binding partner. A“natural binding sequence” may be present either in isolation or in thecontext of a larger polypeptide molecule, which molecule may benaturally-occurring or recombinant. If present, amino acids outside ofthe binding sequence may be either natural, i.e., from the samepolypeptide sequence from which the fragment is derived, or non-natural,i.e., from another (different) polypeptide or from a sequence that isnot derived from any known polypeptide. In assays of the invention, abinding sequence and its binding partner may exist either on twodifferent polypeptide chains or on a single polypeptide chain.

[0048] As used herein, the term “binding polypeptide” refers to amolecule comprising multiple binding sequences, as defined above. Abinding polypeptide of use in the invention is a “natural bindingpolypeptide”, in which the component binding sequences are naturalbinding sequences, as defined above (e.g., wherein the binding sequencesare derived from a single, naturally-occurring polypeptide molecule),and are both necessary and, in combination, sufficient to permitphosphorylation state-dependent binding of the binding polypeptide toits binding partner, wherein the sequences of the binding polypeptideare either contiguous or are non-contiguous. As used herein in referenceto the component binding sequences of a binding polypeptide, the term“non-contiguous” refers to binding sequences which are linked byintervening naturally-occurring, as defined herein, or non-natural aminoacid sequences or other chemical or biological linker molecules such areknown in the art. The amino acids of a polypeptide that do notsignificantly contribute to the natural phosphorylation-state-dependentbinding of that polypeptide to its binding partner may be those aminoacids which are naturally present and link the binding sequences in abinding polypeptide or they may be derived from a different naturalpolypeptide or may be wholly unknown in nature. In assays of theinvention, a binding polypeptide and its binding partner (which may,itself, be a binding domain, sequence or polypeptide, as defined herein)may exist on two different polypeptide chains or on a single polypeptidechain. According to the invention, a natural binding polypeptide, like apolypeptide as defined above, is not a full-length natural polypeptidechain, but instead comprises a subset that encompasses up to one fewerthan the total number of amino acids in a natural polypeptide chain.

[0049] As used herein, the terms “polypeptide” and “peptide” refer to apolymer in which the monomers are amino acids and are joined togetherthrough peptide or disulfide bonds. The terms subunit and domain alsomay refer to polypeptides and peptides having biological function. Apeptide useful in the invention will at least have a binding capability,i.e, with respect to binding as- or to a binding partner, and also mayhave another biological function that is a biological function of aprotein or domain from which the peptide sequence is derived.“Polypeptide” refers to a naturally-occurring amino acid chaincomprising a subset of the amino acids of a full-length protein, whereinthe subset comprises at least one fewer amino acid than does thefull-length protein, or a “fragment thereof” or “peptide”, such as aselected region of the polypeptide that is of interest in a bindingassay and for which a binding partner is known or determinable.“Fragment thereof” thus refers to an amino acid sequence that is aportion of a full-length polypeptide, between about 8 and about 1000amino acids in length, preferably about 8 to about 300, more preferablyabout 8 to about 200 amino acids, and even more preferably about 10 toabout 50 or 100 amino acids in length. “Peptide” refers to a short aminoacid sequence that is 10-40 amino acids long, preferably 10-35 aminoacids. Additionally, unnatural amino acids, for example, β-alanine,phenyl glycine and homoarginine may be included. Commonly-encounteredamino acids which are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-optical isomer. The L-isomers are preferred. Inaddition, other peptidomimetics are also useful, e.g. in linkersequences of polypeptides of the present invention (see Spatola, 1983,in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,Weinstein, ed., Marcel Dekker, New York, p. 267).

[0050] As used herein, the terms “protein”, “subunit” and “domain” referto a linear sequence of amino acids which exhibits biological function.This linear sequence does not include full-length amino acid sequences(e.g. those encoded by a full-length gene or polynucleotide), but doesinclude a portion or fragment thereof, provided the biological functionis maintained by that portion or fragment. The terms “subunit” and“domain” also may refer to polypeptides and peptides having biologicalfunction. A peptide useful in the invention will at least have a bindingcapability, i.e, with respect to binding as or to a binding partner, andalso may have another biological function that is a biological functionof a protein or domain from which the peptide sequence is derived.

[0051] “Polynucleotide” refers to a polymeric form of nucleotides of atleast 10 bases in length and up to 1,000 bases or even more, eitherribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. The term includes single and double stranded formsof DNA.

[0052] Preferably, with regard to the natural binding domain and/orbinding partner therefor, phosphorylation or dephosphorylation isperformed by an enzyme which is a kinase or a phosphatase, respectively.

[0053] It is preferred that phosphorylation of the site prevents bindingof the isolated natural binding domain to the binding partner.

[0054] As used herein, the term “prevents” refers to a reduction of atleast 10%, preferably 20-40%, more preferably 50-75% and, mostpreferably, 80-100% of binding of the isolated natural binding domain tothe binding partner therefor.

[0055] Preferably, phosphorylation of the site promotes binding of theisolated natural binding domain to the binding partner.

[0056] As used herein with regard to protein:protein binding, the term“promotes” refers to that which causes an increase in binding of thenatural binding domain and its binding partner of at least two-fold,preferably 10- to 20-fold, highly preferably 50- to 100-fold, morepreferably from 200- to 1000-fold, and, most preferably, from 200 to10,000-fold.

[0057] It is preferred that dephosphorylation of the site preventsbinding of the isolated natural binding domain to the binding partner.

[0058] It is additionally preferred that dephosphorylation of the sitepromotes binding of the isolated natural binding domain to the bindingpartner.

[0059] In a preferred embodiment, at least one of the isolated naturalbinding domain and the binding partner comprises a detectable label.

[0060] Preferably, the detectable label emits light.

[0061] More preferably, the light is fluorescent.

[0062] It is preferred that one of the isolated natural binding domainand the binding partner therefor comprises a quencher for the detectablelabel. Labels of use in the invention include, but are not limited to, aradioactive label, a fluorescent label and a quencher for either.

[0063] A “fluorescent label”, “fluorescent tag” or “fluorescent group”refers to either a fluorophore or a fluorescent protein or fluorescentfragment thereof.

[0064] “Fluorescent protein” refers to any protein which fluoresces whenexcited with appropriate electromagnetic radiation. This includes aprotein whose amino acid sequence is either natural or engineered. A“fluorescent protein” is a full-length fluorescent protein orfluorescent fragment thereof . By the same token, the term “linker”refers to that which is coupled to both the donor and acceptor proteinmolecules, such as an amino acid sequence joining two natural bindingdomains or a disulfide bond between two polypeptides.

[0065] It is contemplated that with regard to fluorescent labelsemployed in FRET, the reporter labels are chosen such that the emissionwavelength spectrum of one (the “donor”) is within the excitationwavelength spectrum of the other (the “acceptor”). With regard to afluorescent label and a quencher employed in a single-label detectionprocedure in an assay of the invention, it is additionally contemplatedthat the fluorophore and quencher are chosen such that the emissionwavelength spectrum of the fluorophore is within the absorption spectrumof the quencher, such that when the fluorophore and the quencher withwhich it is employed are brought into close proximity by binding of thenatural binding domain, sequence or polypeptide upon which one ispresent with the binding partner comprising the other, detection of thefluorescent signal emitted by the fluorophore is reduced by at least10%, preferably 20-50%, more preferably, 70-90% and, most preferably, by95-100%. A typical quencher reduces detection of a fluorescent signal byapproximately 80%.

[0066] Another aspect of the invention is a kit comprising an isolatednatural binding domain and a binding partner therefor, wherein theisolated natural binding domain includes a site for post-translationalphosphorylation and binds the binding partner in a manner dependent uponphosphorylation of the site, and packaging material therefor.

[0067] It is preferred that the kit further comprises a buffer whichpermits phosphorylation-dependent binding of the isolated naturalbinding domain and the binding partner.

[0068] As used herein, the term “buffer” refers to a medium whichpermits activity of the protein kinase or phosphatase used in an assayof the invention, and is typically a low-ionic-strength buffer or otherbiocompatible solution (e.g., water, containing one or more ofphysiological salt, such as simple saline, and/or a weak buffer, such asTris or phosphate, or others as described hereinbelow), a cell culturemedium, of which many are known in the art, or a whole or fractionatedcell lysate. Such a buffer permits phosphorylation-dependent binding ofa natural binding domain of the invention and a binding partner thereforand, preferably, inhibits degradation and maintains biological activityof the reaction components. Inhibitors of degradation, such as proteaseinhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors(e.g., DEPC) are well known in the art. Lastly, an appropriate buffermay comprise a stabilizing substance such as glycerol, sucrose orpolyethylene glycol.

[0069] As used herein, the term “physiological buffer” refers to aliquid medium that mimics the salt balance and pH of the cytoplasm of acell or of the extracellular milieu, such that post-translationalprotein modification reactions and protein:protein binding are permittedto occur in the buffer as they would in vivo.

[0070] Preferably, the buffer permits phosphorylation ordephosphorylation of the site by a kinase or a phosphatase,respectively.

[0071] In a preferred embodiment, the kit further comprises one or bothof a kinase and a phosphatase.

[0072] It is preferred that the kit further comprises a substrate forthe phosphatase or kinase, the substrate being MGATP.

[0073] It is contemplated that at least a part of a substrate of anenzyme of use in an assay of the invention is transferred to aphosphorylation site on an isolated polypeptide of the invention. Asused herein, the term “at least a part of a substrate” refers to aportion (e.g., a moiety or a group, as defined above) which comprisesless than the whole of the substrate for the enzyme, the transfer ofwhich portion to a phosphorylation site on an isolated polypeptide, bothas defined above, is catalyzed by the enzyme.

[0074] It is additionally preferred that the kit further comprises acofactor for one or both of the kinase or phosphatase. Cofactors of usein the invention include, but are not limited to, cAMP,phosphotidylserine, diolein, Mn²⁺ and Mg²⁺.

[0075] Preferably, at least one of the isolated natural binding domainand the binding partner comprises a detectable label.

[0076] It is preferred that the detectable label emits light, and morepreferred that the light is fluorescent.

[0077] An enzyme (e.g., a protein kinase or phosphatase) of use in theinvention may be natural or recombinant or, alternatively, may bechemically synthesized. If either natural or recombinant, it may besubstantially pure (i.e., present in a population of molecules in whichit is at least 50% homogeneous), partially purified (i.e., representedby at least 1% of the molecules present in a fraction of a cellularlysate) or may be present in a crude biological sample.

[0078] As used herein, the term “sample” refers to a collection ofinorganic, organic or biochemical molecules which is either found innature (e.g., in a biological- or other specimen) or in anartificially-constructed grouping, such as agents which might be foundand/or mixed in a laboratory. Such a sample may be either heterogeneousor homogeneous.

[0079] As used herein, the interchangeable terms “biological specimen”and “biological sample” refer to a whole organism or a subset of itstissues, cells or component parts (e.g. body fluids, including but notlimited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluidand semen). “Biological sample” further refers to a homogenate, lysateor extract prepared from a whole organism or a subset of its tissues,cells or component parts, or a fraction or portion thereof. Lastly,“biological sample” refers to a medium, such as a nutrient broth or gelin which an organism has been propagated, which contains cellularcomponents, such as proteins or nucleic acid molecules.

[0080] As used herein, the term “organism” refers to all cellularlife-forms, such as prokaryotes and eukaryotes, as well as non-cellular,nucleic acid-containing entities, such as bacteriophage and viruses.

[0081] In a method as described above, it is preferred that at least oneof the isolated natural binding domain and the binding partner islabeled with a detectable label.

[0082] Preferably, the label emits light and, more preferably, the lightis fluorescent.

[0083] In another preferred embodiment, the detection step is to detecta change in signal emission by the detectable label.

[0084] It is preferred that the method further comprises exciting thedetectable label and monitoring fluorescence emission.

[0085] It is additionally preferred that the method further comprisesthe step, prior to or after the detection step, of contacting theisolated natural binding domain and the binding partner with an agentwhich modulates the activity of the enzyme.

[0086] As used herein with regard to a biological or chemical agent, theterm “modulate” refers to enhancing or inhibiting the activity of aprotein kinase or phosphatase in an assay of the invention; suchmodulation may be direct (e.g. including, but not limited to, cleavageof- or competitive binding of another substance to the enzyme) orindirect (e.g. by blocking the initial production or, if required,activation of the kinase or phosphatase).

[0087] “Modulation” refers to the capacity to either increase or deceasea measurable functional property of biological activity or process(e.g., enzyme activity or receptor binding) by at least 10%, 15%, 20%,25%, 50%, 100% or more; such increase or decrease may be contingent onthe occurrence of a specific event, such as activation of a signaltransduction pathway, and/or may be manifest only in particular celltypes.

[0088] The term “modulator” refers to a chemical compound (naturallyoccurring or non-naturally occurring), such as a biologicalmacromolecule (e.g., nucleic acid, protein, non-peptide, or organicmolecule), or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues, or even an inorganic element or molecule. Modulators areevaluated for potential activity as inhibitors or activators (directlyor indirectly) of a biological process or processes (e.g., agonist,partial antagonist, partial agonist, antagonist, antineoplastic agents,cytotoxic agents, inhibitors of neoplastic transformation or cellproliferation, cell proliferation-promoting agents, and the like) byinclusion in screening assays described herein. The activities (oractivity) of a modulator may be known, unknown or partially-known. Suchmodulators can be screened using the methods described herein.

[0089] The term “candidate modulator” refers to a compound to be testedby one or more screening method(s) of the invention as a putativemodulator. Usually, various predetermined concentrations are used forscreening such as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as describedmore fully hereinbelow. Test compound controls can include themeasurement of a signal in the absence of the test compound orcomparison to a compound known to modulate the target.

[0090] The invention additionally provides a method of screening for acandidate modulator of enzymatic activity of a kinase or a phosphatase,the method comprising contacting an isolated natural binding domain, abinding partner therefor and an enzyme with a candidate modulator of thekinase or phosphatase, wherein the natural binding domain includes asite for post-translational phosphorylation and binds the bindingpartner in a manner that is dependent upon phosphorylation ordephosphorylation of the site by the kinase or phosphatase and whereinat least one of the isolated natural binding domain and the bindingpartner comprises a detectable label, and monitoring the binding of theisolated natural binding domain to the binding partner, wherein bindingor dissociation of the isolated natural binding domain and the bindingpartner as a result of the contacting is indicative of modulation ofenzymatic activity by the candidate modulator of the kinase orphosphatase.

[0091] Preferably, the detectable label emits light.

[0092] More preferably, the light is fluorescent.

[0093] It is preferred that the monitoring comprises measuring a changein energy transfer between a detectable label present on the isolatednatural binding domain and a detectable label present on the bindingpartner.

[0094] A final aspect of the invention is a method of screening for acandidate modulator of enzymatic activity of a kinase or a phosphatase,the method comprising contacting an assay system with a candidatemodulator of enzymatic activity of a kinase or phosphatase, andmonitoring binding of an isolated natural binding domain and a bindingpartner therefor in the assay system, wherein the isolated naturalbinding domain includes a site for post-translational phosphorilationand binds the binding partner in a manner that is dependent uponphosphorylation or dephosphorylation of the site by a kinase orphosphatase in the assay system, wherein at least one of the isolatednatural binding domain and the binding partner comprises a detectablelabel, and wherein binding or dissociation of the isolated naturalbinding domain and the binding partner as a result of the contacting isindicative of modulation of enzymatic activity by the candidatemodulator of a the kinase or phosphatase.

[0095] It is highly preferred that in any of the above methods, themethod comprises real-time observation of association of an isolatednatural binding domain and its binding partner.

[0096] Further features and advantages of the invention will become morefully apparent in the following description of the embodiments anddrawings thereof, and from the claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0097]FIG. 1 diagrams double- and single-chain enzymatic assay formatsof the invention.

[0098]FIG. 2 presents a schematic overview of FRET in an assay of theinvention.

[0099]FIG. 3 presents monomer:excimer fluorescence.

[0100]FIG. 4 demonstrates the results of FRET between ZAP70-GFP and arhodamine labelled TCRζ derived peptide.

[0101]FIG. 5 demonstrates the dependence of YOP activity on differentconcentrations of TCRζ peptide.

[0102]FIG. 6 presents the results of a FRET based assay for measuringinhibition of YOP by sodium orthovanadate.

[0103]FIG. 7 demonstrates detection of binding of Chk1 phosphorylated,fluorescein labelled Chktide to 14-3-3ζ by fluorescence polarisation.

[0104]FIG. 8 demonstrates inhibition of Chk1 phosphorylation of Chktidepeptide by EDTA.

[0105]FIG. 9 presents the results of a real time assay for Chk1 activitymonitoring the fluorescence polarisation of fluorescein labelled Chktidesubstrate binding to 14-3-3ζ protein.

[0106]FIG. 10 demonstrates that Chk1 activity measured by Chktide:14-3-3 binding is dependent on ATP and the presence of 14-3-3ζ protein.

[0107]FIG. 11 demonstrates inhibition of Chk1 phosphorylation offluorescein labelled Chktide by EDTA.

[0108]FIG. 12 presents the results of an assay for Chk1 phosphorylationof Chktide peptide as measured by 14-3-3∈ binding.

[0109]FIG. 13 demonstrates phosphatase λ activity as measured bydephosphorylation of fluorescein labelled Chktide and decreased bindingto 14-3-3ζ.

[0110]FIG. 14 demonstrates phosphatase λ activity as measured bydephosphorylation of fluorescein labelled Chktide and decreased bindingto 14-3-3∈.

[0111]FIG. 15 presents a time course of Chk1 and PKA activity measuredusing fluorescence polarisation.

[0112]FIG. 16 demonstrates detection of peptide phosphorylation by Srckinase, by measuring FRET between ZAP-GFP and a rhodamine labelledsubstrate peptide.

[0113]FIG. 17 demonstrates detection of SHPS-1 derived peptidephosphorylation by Src, and binding of SHPS-1 to SHP2-GFP partner.

[0114]FIG. 18 demonstrates YOP mediated reversal of FRET betweenSHP2-GFP and rhodamine labelled, phosphorylated, SHPS-1 peptide.

[0115]FIG. 19 presents detection of Src inhibition by staurosporineusing a FRET-based assay between rhodamine labelled SHPS-1 and SHP2-GFP.

[0116]FIG. 20 presents the results of a real-time, FRET-based assay,measuring Src phosphorylation of SHPS-1 peptide.

DESCRIPTION

[0117] The invention is based upon the discovery that a natural bindingdomain, sequence or polypeptide, as defined above, associates with abinding partner to form a complex or dissociates from a binding partner,in a manner that is dependent upon the presence or absence of aphosphate moiety, and that is detectable and measurable in a highlysensitive manner that may be observed in real time.

[0118] Polypeptides of Use in the Invention

[0119] The invention provides reporter molecules and assays formeasuring the activity of protein kinases and phosphatases. Thesereporter molecules are naturally-occurring polypeptides which includenatural binding domains, natural binding sequences and natural bindingpolypeptides, each as defined above, which are used in assays of theinvention in combination with polypeptide binding partners, also asdefined above.

[0120] Minimally, such a reporter molecule comprises or consists of anatural binding domain. The amino acids of a natural binding domain arethose which are necessary for phosphorylation-dependent binding of themolecule comprising or consisting of the natural binding domain with abinding partner, whether such a partner is present on the same or adifferent polypeptide chain as the natural binding domain. Such aminoacids may include points of direct contact between the domain and thebinding partner, those which are recognized and/or modified (i.e.,phosphorylated or dephosphorylated) by a kinase or phosphatase and thosewhich maintain the three-dimensional structure or charge of the bindingdomain in a manner which permits phosphorylation and/ordephosphorylation and the consequent phosphorylation- and/ordephosphorylation-dependent binding of the domain to the bindingpartner. The amino acids of a natural binding domain may be contiguousor may be separated by non-domain amino acids; such non-domain residuesmay be either those which are naturally present between the amino acidsof the natural binding domain or which are non-natural. In cases inwhich non-natural amino acids are found interspersed with those of anatural binding domain, such non-natural residues will be residues whichdo not substantially (that is, measurably) alter the naturalphosphorylation-dependent binding of the natural binding domain to itsbinding partner.

[0121] A second reporter molecule of use in the invention is that whichcomprises or consists of a naturally-occurring stretch of contiguousamino acids sufficient for phosphorylation-dependent binding to abinding partner, as defined above, i.e., at least the minimum number ofcontiguous amino acids required to encompass a natural binding domain.The phosphorylation-dependence of such a molecule, referred to herein asa “natural binding sequence”, is, itself natural. A reporter molecule ofthe invention may either consist of or comprise a natural bindingsequence. In the latter case, amino acids outside of the natural bindingsequence do not substantially influence phosphorylation-dependentbinding of the natural binding domain to the binding partner.

[0122] Lastly, a reporter molecule of use in the invention may be a“natural binding polypeptide”, as defined above. Such a polypeptidemolecule comprises or consists of multiple natural binding domains(above), which domains are, either individually or in concert with oneanother, sufficient to permit natural, phosphorylation-dependent bindingof the natural binding polypeptide to a binding partner.

[0123] By monitoring the association or dissociation of a naturalbinding domain, sequence or polypeptide and its binding partner in thepresence of a known or candidate protein kinase or phosphatase, theactivity of such an enzyme can be measured. In such assays, one or bothof the natural binding domain, sequence or polypeptide and its bindingpartner comprises a detectable label including, but not exclusively, afluorescent or other light-emitting label, which may be either chemicalor proteinaceous. By measuring changes in signal emission or absorptionbefore and after addition to the mixture comprising the natural bindingdomain, sequence or polypeptide and its binding partner of the enzyme tobe assayed, the extent of phosphorylation can be calculated. Animportant feature of the invention is that such measurements (e.g., of ashift in FRET) can be performed in real-time. This allows for sensitiveassessment of enzyme reaction kinetics based upon the rate of change ofthe protein-binding-dependent signal emission or absorption by thelabel(s).

[0124] Assays in which the above reporter molecules are used accordingto the invention may be performed either in double- or single-chainformat (FIG. 1). In double-chain format, natural binding domain,sequence or polypeptide is comprised by a different polypeptide chainfrom that comprising or consisting of the binding partner and is nototherwise covalently linked to it. In single-chain format, the naturalbinding domain, sequence or polypeptide is covalently linked to itsbinding partner, either through an intervening amino acid sequence or achemical linker.

[0125] The binding partner of a natural binding domain, sequence orpolypeptide may, itself, be a natural binding domain, sequence orpolypeptide as defined herein. If so, binding of the two molecules maydepend upon the phosphorylation state of one or both in a manner that iscomparable to that found in nature.

[0126] Methods by which assays of the invention are performed aredescribed in detail in the following sections and in the Examples.

[0127] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art (e.g, in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., which is incorporated herein by reference), chemical methods,pharmaceutical formulation and delivery and treatment of patients.

[0128] Methods by which to Detect Protein:Protein Binding in Assays ofthe Invention

[0129] Methods of detecting the phosphorylation-dependent binding of anatural binding domain, sequence or polypeptide and a binding partner inan assay of the invention most usefully, although not exclusively, arethose which employ light-emitting labels. Several such techniques aredescribed below.

[0130] Fluorescence Energy Resonance Transfer (FRET)

[0131] A tool with which to assess the distance between one molecule andanother (whether protein or nucleic acid) or between two positions onthe same molecule is provided by the technique of fluorescence resonanceenergy transfer (FRET), which is now widely known in the art (for areview, see Matyus, 1992, J. Photochem. Photobiol. B: Biol., 12:323-337, which is herein incorporated by reference). FRET is aradiationless process in which energy is transferred from an exciteddonor molecule to an acceptor molecule; the efficiency of this transferis dependent upon the distance between the donor an acceptor molecules,as described below. Since the rate of energy transfer is inverselyproportional to the sixth power of the distance between the donor andacceptor, the energy transfer efficiency is extremely sensitive todistance changes. Energy transfer is said to occur with detectableefficiency in the 1-10 nm distance range, but is typically 4-6 nm forfavorable pairs of donor and acceptor.

[0132] Radiationless energy transfer is based on the biophysicalproperties of fluorophores. These principles are reviewed elsewhere(Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press,New York; Jovin and Jovin, 1989, Cell Structure and Function byMicrospectrofluorometry, eds. E. Kohen and J. G. Hirschberg, AcademicPress, both of which are incorporated herein by reference). Briefly, afluorophore absorbs light energy at a characteristic wavelength. Thiswavelength is also known as the excitation wavelength. The energyabsorbed by a flurochrome is subsequently released through variouspathways, one being emission of photons to produce fluorescence. Thewavelength of light being emitted is known as the emission wavelengthand is an inherent characteristic of a particular fluorophore.Radiationless energy transfer is the quantum-mechanical process by whichthe energy of the excited state of one fluorophore is transferredwithout actual photon emission to a second fluorophore. That energy maythen be subsequently released at the emission wavelength of the secondfluorophore. The first fluorophore is generally termed the donor (D) andhas an excited state of higher energy than that of the secondfluorophore, termed the acceptor (A). The essential features of theprocess are that the emission spectrum of the donor overlap with theexcitation spectrum of the acceptor, and that the donor and acceptor besufficiently close. The distance over which radiationless energytransfer is effective depends on many factors including the fluorescencequantum efficiency of the donor, the extinction coefficient of theacceptor, the degree of overlap of their respective spectra, therefractive index of the medium, and the relative orientation of thetransition moments of the two fluorophores. In addition to having anoptimum emission range overlapping the excitation wavelength of theother fluorophore, the distance between D and A must be sufficientlysmall to allow the radiationless transfer of energy between thefluorophores.

[0133] FRET may be performed either in vivo or in vitro. Proteins arelabeled either in vivo or in vitro by methods known in the art.According to the invention, a natural binding domain, sequence orpolypeptide and its binding partner, comprised either by the same or bydifferent polypeptide molecules, are differentially labeled, one with adonor and the other with an acceptor, and differences in fluorescencebetween a test assay, comprising a protein modifying enzyme, and acontrol, in which the modifying enzyme is absent, are measured using afluorimeter or laser-scanning microscope. It will be apparent to thoseskilled in the art that excitation/detection means can be augmented bythe incorporation of photomultiplier means to enhance detectionsensitivity. The differential labels may comprise either two differentfluorescent labels (e.g., fluorescent proteins as described below or thefluorophores rhodamine, fluorescein, SPQ, and others as are known in theart) or a fluorescent label and a molecule known to quench its signal;differences in the proximity of the natural binding domain, sequence orpolypeptide with its binding partner with and without theprotein-modifying enzyme can be gauged based upon a difference in thefluorescence spectrum or intensity observed.

[0134] This combination of protein-labeling methods and devices confersa distinct advantage over prior art methods for determining the activityof protein-modifying enzymes, as described above, in that results of allmeasurements are observed in real time (i.e., as a reaction progresses).This is significantly advantageous, as it allows both for rapid datacollection and yields information regarding reaction kinetics undervarious conditions.

[0135] A sample, whether in vitro or in vivo, assayed according to theinvention therefore comprises a mixture at equilibrium of the labelednatural binding domain, sequence or polypeptide and its binding partnerwhich, when disassociated from one another, fluoresce at one frequencyand, when complexed together, fluoresce at another frequency or,alternatively, of molecules which either do or do not fluoresce or showreduced fluorescence, depending upon whether or not they are associated.

[0136] The natural binding domain, sequence or polypeptide is modifiedto allow the attachment of a fluorescent label to the surface of thatmolecule or is fused in-frame with a fluorescent protein, as describedbelow. The choice of fluorescent label will be such that upon excitationwith light, labeled peptides which are associated will show optimalenergy transfer between fluorophores. In the presence of a proteinkinase or phosphatase, the natural binding domain, sequence orpolypeptide and its binding partner dissociate due to a structural orelectrostatic change which occurs as a consequence of addition orremoval of a phosphate to/from the enzyme recognition site, therebyleading to a decrease in energy transfer and increased emission of lightby the donor fluorophore. In this way, the state of polypeptidephosphorylation can be monitored and quantitated in real-time. Thisscheme, which represents the broadest embodiment of the invention, isshown in FIG. 2.

[0137] As used herein, the terms “fluorophore” and “fluorochrome” referinterchangeably to a molecule which is capable of absorbing energy at awavelength range and releasing energy at a wavelength range other thanthe absorbance range. The term “excitation wavelength” refers to therange of wavelengths at which a fluorophore absorbs energy. The term“emission wavelength” refers to the range of wavelength that thefluorophore releases energy or fluoresces.

[0138] A non-limiting list of chemical fluorophores of use in theinvention, along with their excitation and emission wavelengths, ispresented in Table 1. TABLE 1 Fluorophore Excitation (nm) Emission (nm)Color PKH2 490 504 green PKH67 490 502 green Fluorescein (FITC) 495 525green Hoechst 33258 360 470 blue R-Phycoerythrin (PE) 488 578 orange-redRhodamine (TRITC) 552 570 red Quantum Red ™ 488 670 red PKH26 551 567red Texas Red 596 620 red Cy3 552 570 red

[0139] Examples of fluorescent proteins which vary among themselves inexcitation and emission maxima are listed in Table 1 of WO 97/28261(Tsien et al., 1997, supra). These (each followed by [excitationmax./emission max.] wavelengths expressed in nanometers) includewild-type Green Fluorescent Protein [395(475)/508] and the cloned mutantof Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7[433(453)/475(501)], W2 [432(453)/480], S65T [489/511], P4-1[504(396)/480], S65A [471/504], S65C [479/507], S65L [484/510], Y66F[360/442], Y66W [458/480], I0c [513/527],W1B [432(453)/476(503)],Emerald [487/508] and Sapphire [395/511]. This list is not exhaustive offluorescent proteins known in the art; additional examples are found inthe Genbank and SwissProt public databases.

[0140] A number of parameters of fluorescence output are envisagedincluding

[0141] 1) measuring fluoresence emitted at the emission wavelength ofthe acceptor (A) and donor (D) and determining the extent of energytransfer by the ratio of their emission amplitudes;

[0142] 2) measuring the fluoresence lifetime of D;

[0143] 3) measuring the rate of photobleaching of D;

[0144] 4) measuring the anisotropy of D and/or A; or

[0145] 5) measuring the Stokes shift monomer; excimer fluorescence.

[0146] Certain of these techniques are presented below.

[0147] Alternative Fluorescent Techniques Suitable for MonitoringProtein:Protein Binding in Assays of the Invention

[0148] One embodiment of the technology can utilize monomer:excimerfluorescence as the output. The association of a natural binding domainwith a binding partner in this format is shown in FIG. 3.

[0149] The fluorophore pyrene when present as a single copy displaysfluorescent emission of a particular wavelength significantly shorterthan when two copies of pyrene form a planar dimer (excimer), asdepicted. As above, excitation at a single wavelength (probably 340 nm)is used to review the excimer fluorescence (˜470 nm) over monomerfluorescence (˜375 nm) to quantify assembly:disassembly of the reportermolecule.

[0150] Additional embodiments of the present invention are not dependenton FRET. For example the invention can make use of fluorescencecorrelation spectroscopy (FCS), which relies on the measurement of therate of diffusion of a label (see Elson and Magde, 1974 Biopolymers, 13:1-27; Rigler et al., 1992, in Fluorescence Spectroscopy: New Methods andApplications, Springer Verlag, pp.13-24; Eigen and Rigler, 1994, Proc.Natl. Acad. Sci. U.S.A., 91: 5740-5747; Kinjo and Rigler, 1995, NucleicAcids Res., 23: 1795-1799).

[0151] In FCS, a focused laser beam illuminates a very small volume ofsolution, of the order of 10⁻¹⁵ liter, which at any given point in timecontains only one molecule of the many under analysis. The diffusion ofsingle molecules through the illuminated volume, over time, results inbursts of fluorescent light as the labels of the molecules are excitedby the laser. Each individual burst, resulting from a single molecule,can be registered.

[0152] A labeled polypeptide will diffuse at a slower rate if it islarge than if it is small. Thus, multimerized polypeptides will displayslow diffusion rates, resulting in a lower number of fluorescent burstsin any given timeframe, while labeled polypeptides which are notmultimerized or which have dissociated from a multimer will diffuse morerapidly. Binding of polypeptides according to the invention can becalculated directly from the diffusion rates through the illuminatedvolume.

[0153] Where FCS is employed, rather than FRET, it is not necessary tolabel more than one polypeptide. Preferably, a single polypeptide memberof the multimer is labeled. The labeled polypeptide dissociates from themultimer as a result of modification, thus altering the FCS reading forthe fluorescent label.

[0154] A further detection technique which may be employed in the methodof the present invention is the measurement of time-dependent decay offluorescence anisotropy. This is described, for example, in Lacowicz,1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York,incorporated herein by reference (see, for example, page 167).

[0155] Fluorescence anisotropy relies on the measurement of the rotationof fluorescent groups. Larger multimers of polypeptides rotate moreslowly than monomers, allowing the formation of multimers to bemonitored.

[0156] Non-Fluorescent Detection Methods for Use in the Invention

[0157] The invention may be configured to exploit a number ofnon-fluorescent labels. In a first embodiment, the natural bindingdomain and binding partner therefor form, when bound, an active enzymewhich is capable of participating in an enzyme-substrate reaction whichhas a detectable endpoint. The enzyme may comprise two or morepolypeptide chains or regions of a single chain, such that upon bindingof the natural binding domain to the binding partner, which are presenteither on two different polypeptide chains or in two different regionsof a single polypeptide, these components assemble to form a functionalenzyme. Enzyme function may be assessed by a number of methods,including scintillation counting and photospectroscopy. In a furtherembodiment, the invention may be configured such that the label is aredox enzyme, for example glucose oxidase, and the signal generated bythe label is an electrical signal.

[0158] Phosphorylation of the natural binding domain and, optionally,its binding partner according to the invention is required to inhibitbinding and, consequently, enzyme component assembly, thus reducingenzyme activity.

[0159] In another assay format, an enzyme is used together with amodulator of enzyme activity, such as an inhibitor or a cofactor. Insuch an assay, one of the enzyme and the inhibitor or cofactor is annatural binding domain, the other its binding partner. Binding of theenzyme to its inhibitor or cofactor results in modulation of enzymaticactivity, which is detectable by conventional means (such as monitoringfor the conversion of substrate to product for a given enzyme).

[0160] Fluorescent Protein Labels in Assays of the Invention

[0161] In a FRET assay of the invention, the fluorescent protein labelsare chosen such that the excitation spectrum of one of the labels (theacceptor) overlaps with the emission spectrum of the excited fluorescentlabel (the donor). The donor label is excited by light of appropriateintensity within the donor's excitation spectrum. The donor then emitssome of the absorbed energy as fluorescent light and dissipates some ofthe energy by FRET to the acceptor fluorescent label. The fluorescentenergy it produces is quenched by the acceptor fluorescent proteinlabel. FRET can be manifested as a reduction in the intensity of thefluorescent signal from the donor, reduction in the lifetime of itsexcited state, and re-emission of fluorescent light at the longerwavelengths (lower energies) characteristic of the acceptor. When thedonor and acceptor labels become spatially separated, FRET is diminishedor eliminated.

[0162] One can take advantage of the FRET exhibited by a natural bindingdomain, sequence or polypeptide and its binding partner labeled withdifferent fluorescent proteins, wherein one is linked to a donor and theother to an acceptor fluorescent protein, in monitoring proteinphosphorylation according to the present invention. A single polypeptidemay comprises a blue fluorescent protein donor and a green fluorescentprotein acceptor, wherein each is fused to a different assay component(i.e., in which one is fused to the natural binding domain, sequence orpolypeptide and the other to its binding partner); such a construct isherein referred to as a “tandem” fusion protein. Alternatively, twodistinct polypeptides (“single” fusion proteins) one comprising anatural binding domain, sequence or polypeptide and the other itsbinding partner may be differentially labeled with the donor andacceptor fluorescent proteins, respectively. The construction and use oftandem fusion proteins in the invention can reduce significantly themolar concentration of peptides necessary to effect an associationbetween differentially-labeled polypeptide assay components relative tothat required when single fusion proteins are instead used. The labelednatural binding domain, sequence or polypeptide and/or its bindingpartner may be produced via the expression of recombinant nucleic acidmolecules comprising an in-frame fusion of sequences encoding a such apolypeptide and a fluorescent protein label either in vitro (e.g., usinga cell-free transcription/translation system, as described below, orinstead using cultured cells transformed or transfected using methodswell known in the art) or in vivo, for example in a transgenic animalincluding, but not limited to, insects, amphibians and mammals. Arecombinant nucleic acid molecule of use in the invention may beconstructed and expressed by molecular methods well known in the art,and may additionally comprise sequences including, but not limited to,those which encode a tag (e.g., a histidine tag) to enable easypurification, a secretion signal, a nuclear localization signal or otherprimary sequence signal capable of targeting the construct to aparticular cellular location, if it is so desired.

[0163] The means by which a natural binding domain, sequence orpolypeptide and its binding partner are assayed for association usingfluorescent protein labels according to the invention may be brieflysummarized as follows:

[0164] Whether or not the natural binding domain, sequence orpolypeptide and its binding partner are present on a single polypeptidemolecule, one is labeled with a green fluorescent protein, while theother is preferably labeled with a red or, alternatively, a bluefluorescent protein. Useful donor:acceptor pairs of fluorescent proteins(see Tsien et al., 1997, supra) include, but are not limited to:

[0165] Donor: S72A, K79R, Y145F, M153A and T203I (excitation λ395 nm;emission λ511)

[0166] Acceptor: S65G, S72A, K79R and T203Y (excitation λ514 nm;emission λ527 nm), or

[0167] T203Y/S65G, V68L, Q69K or S72A (excitation λ515 nm; emission λ527nm).

[0168] An example of a blue:green pairing is P4-3 (shown in Table 1 ofTsien et al., 1997, supra) as the donor label and S65C (also of Table 1of Tsien et al., 1997, supra) as the acceptor label. The natural bindingdomain, sequence or polypeptide and corresponding binding partner areexposed to light at, for example, 368 nm, a wavelength that is near theexcitation maximum of P4-3. This wavelength excites S65C only minimally.Upon excitation, some portion of the energy absorbed by the bluefluorescent protein donor is transferred to the acceptor through FRET ifthe natural binding domain, sequence or polypeptide and its bindingpartner are in close association. As a result of this quenching, theblue fluorescent light emitted by the blue fluorescent protein is lessbright than would be expected if the blue fluorescent protein existed inisolation. The acceptor (S65C) may re-emit the energy at longerwavelength, in this case, green fluorescent light.

[0169] After phosphorylation or dephosphorylation of one or both of thenatural binding domain, sequence or polypeptide and its binding partnerby an kinase or phosphatase, respectively, the natural binding domain,sequence or polypeptide and its binding partner (and, hence, the greenand red or, less preferably, green and blue fluorescent proteins)physically separate or associate, accordingly inhibiting or promotingFRET. For example, if activity of the modifying enzyme results indissociation of a protein:protein dimer, the intensity of visible bluefluorescent light emitted by the blue fluorescent protein increases,while the intensity of visible green light emitted by the greenfluorescent protein as a result of FRET, decreases.

[0170] Such a system is useful to monitor the activity of enzymes thatphosphorylate or dephosphorylate the phosphorylation site of a naturalbinding domain, sequence or polypeptide and, optionally, its bindingpartner to which the fluorescent protein labels are fused, as well asthe activity of kinases or phosphatases or candidate modulators of thoseenzymes.

[0171] In particular, this invention contemplates assays in which theamount- or activity of a modifying enzyme in a sample is determined bycontacting the sample with a natural binding domain, sequence orpolypeptide and its binding partner, differentially-labeled withfluorescent proteins, as described above, and measuring changes influorescence of the donor label, the acceptor label or the relativefluorescence of both. Fusion proteins, as described above, whichcomprise either one or both of the labeled natural binding domain,sequence or polypeptide and its binding partner of an assay of theinvention can be used for, among other things, monitoring the activityof a protein kinase or phosphatase inside the cell that expresses therecombinant tandem construct or two different recombinant constructs.

[0172] Advantages of single- and tandem fluorescent protein/polypeptidescomprising a natural binding domain, sequence or polypeptide fused to afluorescent protein include the potential to express the natural bindingdomain, sequence or polypeptide in the cell (providing a convenientexperimental format), the greater extinction coefficient and quantumyield of many of these proteins compared with those of the Edansfluorophore. Also, the acceptor in such a construct or pair ofconstructs is, itself, a fluorophore rather than a non-fluorescentquencher like Dabcyl. Alternatively, in single-label assays of theinvention, whether involving use of a chemical fluorophore or a singlefluorescent fusion construct, such a non-fluorescent quencher may beused. Thus, the enzyme's substrate (i.e., the natural binding domainand, optionally, the corresponding binding partner), and reactionproducts (i.e., the natural binding domain and, optionally, thecorresponding binding partner after modification) are both fluorescentbut with different fluorescent characteristics.

[0173] In particular, the substrate and modified products exhibitdifferent ratios between the amount of light emitted by the donor andacceptor labels. Therefore, the ratio between the two fluorescencesmeasures the degree of conversion of substrate to products, independentof the absolute amount of either, the optical thickness of the sample,the brightness of the excitation lamp, the sensitivity of the detector,etc. Furthermore, Aequorea-derived or -related fluorescent proteinlabels tend to be protease resistant. Therefore, they are likely toretain their fluorescent properties throughout the course of anexperiment.

[0174] Reporter Polypeptide Fusion Construct According to the Invention

[0175] As stated above, recombinant nucleic acid constructs ofparticular use in the invention are those which comprise in-framefusions of sequences encoding a natural binding domain, sequence orpolypeptide or a binding partner therefor and a fluorescent protein. Ifa natural binding domain, sequence or polypeptide and its bindingpartner are to be expressed as part of a single polypeptide, the nucleicacid molecule additionally encodes, at a minimum, a donor fluorescentprotein fused to one, an acceptor fluorescent protein label fused to theother, a linker that couples the two and is of sufficient length andflexibility to allow for folding of the polypeptide and pairing of thenatural binding domain, sequence or polypeptide with the bindingpartner, and gene regulatory sequences operatively linked to the fusioncoding sequence. If single fusion proteins are instead encoded (whetherby one or more nucleic acid molecules), each nucleic acid molecule needonly encode a natural binding domain, sequence or polypeptide or abinding partner therefor, fused either to a donor or acceptorfluorescent protein label and operatively linked to gene regulatorysequences.

[0176] “Operatively-linked” refers to polynucleotide sequences which arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

[0177] As described above, the donor fluorescent protein label iscapable of absorbing a photon and transferring energy to anotherfluorescent label. The acceptor fluorescent protein label is capable ofabsorbing energy and emitting a photon. If needed, the linker connectsthe natural binding domain, sequence or polypeptide and its bindingpartner either directly or indirectly, through an intermediary linkagewith one or both of the donor and acceptor fluorescent protein labels.Regardless of the relative order of the natural binding domain, sequenceor polypeptide, its binding partner and the donor and acceptorfluorescent protein labels on a polypeptide molecule, it is essentialthat sufficient distance be placed between the donor and acceptor by thelinker and/or the natural binding domain, sequence or polypeptide andits binding partner to ensure that FRET does not occur unless thenatural binding domain, sequence or polypeptide and its binding partnerbind. It is desirable, as described in greater detail in WO97/28261, toselect a donor fluorescent protein with an emission spectrum thatoverlaps with the excitation spectrum of an acceptor fluorescentprotein. In some embodiments of the invention the overlap in emissionand excitation spectra will facilitate FRET. Such an overlap is notnecessary, however, if intrinsic fluorescence is measured instead ofFRET. A fluorescent protein of use in the invention includes, inaddition to those with intrinsic fluorescent properties, proteins thatfluoresce due intramolecular rearrangements or the addition of cofactorsthat promote fluorescence.

[0178] For example, green fluorescent proteins (“GFPs”) of cnidarians,which act as their energy-transfer acceptors in bioluminescence, can beused in the invention. A green fluorescent protein, as used herein, is aprotein that fluoresces green light, and a blue fluorescent protein is aprotein that fluoresces blue light. GFPs have been isolated from thePacific Northwest jellyfish, Aequorea victoria, from the sea pansy,Renilla reniformis, and from Phialidium gregarium. (Ward et al., 1982,Photochem. Photobiol., 35: 803-808; Levine et al., 1982, Comp. Biochem.Physiol.,72B: 77-85).

[0179] A variety of Aequorea-related GFPs having useful excitation andemission spectra have been engineered by modifying the amino acidsequence of a naturally occurring GFP from Aequorea victoria. (Prasheret al., 1992, Gene, 111: 229-233; Heim et al., 1994, Proc. Natl. Acad.Sci. U.S.A., 91: 12501-12504; PCT/US95/14692). As used herein, afluorescent protein is an Aequorea-related fluorescent protein if anycontiguous sequence of 150 amino acids of the fluorescent protein has atleast 85% sequence identity with an amino acid sequence, eithercontiguous or non-contiguous, from the wild-type Aequorea greenfluorescent protein of SwissProt Accession No. P42212. Similarly, thefluorescent protein may be related to Renilla or Phialidium wild-typefluorescent proteins using the same standards.

[0180] Aequorea-related fluorescent proteins include, for example,wild-type (native) Aequorea victoria GFP, whose nucleotide and deducedamino acid sequences are presented in Genbank Accession Nos. L29345,M62654, M62653 and others Aequorea-related, engineered versions of GreenFluorescent Protein, of which some are listed above. Several of these,i.e., P4, P4-3, W7 and W2 fluoresce at a distinctly shorter wavelengththan wild type.

[0181] Recombinant nucleic acid molecules encoding single- or tandemfluorescent protein/polypeptide comprising a natural binding domain,sequence or polypeptide or a binding partner therefor fused to afluorescent protein useful in the invention may be expressed for in vivoassay of the activity of a modifying enzyme on the encoded products.Alternatively, the encoded fusion proteins may be isolated prior toassay, and instead assayed in a cell-free in vitro assay system, asdescribed elsewhere herein.

[0182] Protein Phoshorylation in Assays of the Invention

[0183] As highlighted in the Background, the phosphorylation of proteinsis a frequent and important post-translational modification of proteins.There are many examples of situations in which dysfunction of thekinases and phosphatases mediating the phosphorylation state of proteinscan lead to disease. The methods currently available to analyze thephosphorylation state each have drawbacks, as described above. Assayformats of the invention, as outlined in the following sections and inthe Examples, below, will allow monitoring of the phosphorylation stateof a specific target protein or activity of a specific kinase orphosphatase in real time in the cell.

[0184] Three systems, presented in Examples 1 through 4, can be used toexemplify in non-limiting fashion the phosphorylation assay, each ofwhich involves the interaction between a binding domain, sequence orpolypeptide and a binding partner, of which at least the formercomprises a modification site that serves as a substrate for the proteinkinases and phosphatases involved in the system. At the present time,good structural information is available for such interactions.

[0185] Methods for Detection of Protein Phosphorylation in Real Time

[0186] A. In Vitro Protein Modification and Detection thereof

[0187] Modifying Enzymes

[0188] The invention requires the presence of a modifying enzyme whichcatalyzes either the addition or removal of a modifying group. A rangeof kinases, phosphatases and other modifying enzymes are availablecommercially (e.g. from Sigma, St. Louis, Mo.; Promega, Madison, Wis.;Boehringer Mannheim Biochemicals, Indianapolis, Ind.; New EnglandBiolabs, Beverly, Mass.; and others). Alternatively, such enzymes may beprepared in the laboratory by methods well known in the art.

[0189] The catalytic sub-unit of protein kinase A (c-PKA) can bepurified from natural sources (e.g. bovine heart) or fromcells/organisms engineered to heterologously express the enzyme. Otherisoforms of this enzyme may be obtained by these procedures.Purification is performed as previously described from bovine heart(Peters et al.,1977, Biochemistry, 16: 5691-5697) or from a heterologoussource (Tsien et al., WO92/00388), and is in each case brieflysummarized as follows:

[0190] Bovine ventricular cardiac muscle (2 kg) is homogenized and thencentrifuged. The supernatant is applied to a strong anion exchange resin(e.g. Q resin, Bio-Rad) equilibrated in a buffer containing 50 mMTris-HCl, 10 mM NaCl, 4 mM EDTA pH 7.6 and 0.2 mM 2-mercaptoethanol. Theprotein is eluted from the resin in a second buffer containing 50 mMTris-HCl, 4 mM EDTA pH 7.6, 0.2 mM 2-mercaptoethanol, 0.5M NaCl.Fractions containing PKA are pooled and ammonium sulphate added to 30%saturation. Proteins precipitated by this are removed by centrifugationand the ammonium sulphate concentration of the supernatant was increasedto 75% saturation. Insoluble proteins are collected by centrifugation(included c-PKA) and are dissolved in 30 mM phosphate buffer pH 7.0, 1mM EDTA, 0.2 mM 2-mercaptoethanol. These proteins are then dialysedagainst the same buffer (500 volume excess) at 4° C. for two periods of8 hours each. The pH of the sample is reduced to 6.1 by addition ofphosphoric acid, and the sample is mixed sequentially with 5 batches ofCM-Sepharose (Pharmacia, ˜80 ml resin each) equilibrated in 30 mMphosphate pH 6.1, 1 mM EDTA, 0.2 mM 2-mercaptoethanol. Cyclic AMP (10μM) is added to the material which fails to bind to the CM-Sepharose,and the sample-cAMP mix is incubated with a fresh resin of CM-Sepharose(˜100 ml) equilibrated as before. c-PKA is eluted from this columnfollowing extensive washing in equilibration buffer by addition of 30 mMphosphate pH 6.1, 1 mM EDTA, 1M KCl, 0.2 mM 2-mercaptoethanol. Fractionscontaining c-PKA are pooled and concentrated by filtration through aPM-30 membrane (or similar). The c-PKA sample is then subjected togel-filtration chromatography on a resin such as Sephacryl 200HR(Pharmacia).

[0191] The purification of recombinant c-PKA is as described in WO92/00388. General methods of preparing pure and partially-purifiedrecombinant proteins, as well as crude cellular extracts comprising suchproteins, are well known in the art. Molecular methods useful in theproduction of recombinant proteins, whether such proteins are theenzymes to be assayed according to the invention or the labeled reporterpolypeptides of the invention (i.e., the natural binding domain,sequence or polypeptide and its binding partner), are well known in theart (for methods of cloning, expression of cloned genes and proteinpurification, see Sambrook et al., 1989, Molecular Cloning. A LaboratoryManual., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel et al., Current Protocols in Molecular Biology,copyright 1987-1994, Current Protocols, copyright 1994-1998, John Wiley& Sons, Inc.). The sequences of the catalytic subunit of several PKAmolecules are found in the Genbank database (see PKA Cα, bovine, GenbankAccession Nos. X67154 and S49260; PKA Cβ1, bovine, Genbank Accession No.J02647; PKA Cβ2, bovine, M60482, the form most likely purified frombovine heart by the protocol described above).

[0192] According to the invention, assays of the activity of proteinkinases or phosphatases may be performed using crude cellular extracts,whether to test the activity of a recombinant protein or one which isfound in nature, such as in a biological sample obtained from a testcell line or animal or from a clinical patient. In the former case, useof a crude cell extract enables rapid screening of many samples, whichpotentially finds special application in high-throughput screeningmethods, e.g. of candidate modulators of protein kinase/phosphataseactivity. In the latter case, use of a crude extract with the labeledreporter polypeptide comprising a natural binding domain, sequence orpolypeptide of the invention facilitates easy and rapid assessment ofthe activity of an enzyme of interest in a diagnostic procedure, e.g.,one which is directed at determining whether a protein kinase orphosphatase is active at an a physiologically-appropriate level, or in aprocedure designed to assess the efficacy of a therapy aimed atmodulating the activity of a particular enzyme.

[0193] Production of a Natural Binding Domain, Sequence or Polypeptide

[0194] Polypeptides comprising or consisting of a natural bindingdomain, sequence or polypeptide or a binding partner thereof may besynthesized by Fmoc or Tboc chemistry according to methods known in theart (e.g., see Atherton et al., 1981, J. Chem. Soc. Perkin I, 1981(2):538-546; Merrifield, 1963, J. Am. Chem. Soc., 85: 2149-2154,respectively). Following deprotection and cleavage from the resin,peptides are desalted by gel filtration chromatography and analysed bymass spectroscopy, HPLC, Edman degradation and/or other methods as areknown in the art for protein sequencing using standard methodologies.

[0195] Alternatively, nucleic acid sequences encoding such peptides maybe expressed either in cells or in an in vitro transcription/translationsystem (see below) and, as with enzymes to be assayed according to theinvention, the proteins purified by methods well known in the art.

[0196] Labelling of Polypeptides with Fluorophores

[0197] Polypeptides comprising or consisting of natural binding domains,sequences or polypeptides or a binding partner therefor are labeled withthiol reactive derivatives of fluorescein and tetramethylrhodamine(isothiocyanate or iodoacetamide derivatives, Molecular Probes, Eugene,Oreg., USA) or other fluorophores as are known in the art usingprocedures described by Hermanson G. T., 1995, Bioconjugate Techniques,Academic Press, London. Alternatively, primary-amine-directedconjugation reactions can be used to label lysine sidechains or the freepeptide N-terminus (Hermason, 1995, supra).

[0198] Purification of Fluorescent Natural Binding Domains and/orBinding Partners therefor

[0199] Fluorescent peptides are separated from unreacted fluorophores bygel filtration chromatography or reverse phase HPLC.

[0200] Phosphorylation of Natural Binding Domains and, Optionally,Binding Partners therefor In Vitro

[0201] Natural binding domains and, optionally, binding partnerstherefor (0.01-100.0 μM) are phosphorylated by purified c-PKA in 50 mMHistidine buffer pH 7.0, 5 mM MgSO₄, 1 mM EGTA, 0.1-10.0 μM c-PKA, and0.2 mM [³²P] γ-ATP (specific activity ˜2 Bq/pmol) at 15-40° C. forperiods of time ranging from 0 to 60 minutes. Where the chemistry of thepeptide is appropriate (i.e. having a basic charge) the phosphopeptideis captured on a cation exchange filter paper (e.g. phosphocellulose P81paper; Whatman), and reactants are removed by extensive washing in 1%phosphoric acid (see Casnellie, 1991, Methods Enzymol., 200: 115-120).Alternatively, phosphorylation of samples is terminated by the additionof SDS-sample buffer (Laemmli,1970, Nature, 227: 680-685) and thesamples analysed by SDS-PAGE electrophoresis, autoradiography andscintillation counting of gel pieces.

[0202] Dephosphorylation of a Natural Binding Domain or Binding Partnertherefor In Vitro

[0203] The dephosphorylation of natural binding domains and, optionally,binding partners therefor, phosphorylated as above is studied by removalof ATP (through the addition of 10 mM glucose and 30 U/ml hexokinase;Sigma, St. Louis, Mo.) and addition of protein phosphatase-1 (Sigma).Dephosphorylation is followed at 15-40° C. by quantitation of theremaining phosphopeptide component at various time points, determined asabove.

[0204] Fluorescence Measurements of Protein Modification In Vitro inReal Time

[0205] Donor and acceptor fluorophore-labeled polypeptides comprising orconsisting of natural binding domains, sequences or polypeptides (molarequivalents of fluorophore-labeled polypeptide or molar excess ofacceptor-labeled polypeptide) are first mixed (if the natural bindingdomains, sequence or polypeptide and its binding partner are present onseparate polypeptides). Samples are analyzed in a fluorimeter usingexcitation wavelengths relevant to the donor fluorescent label andemission wavelengths relevant to both the donor and acceptor labels. Aratio of emission from the acceptor over that from the donor followingexcitation at a single wavelength is used to determine the efficiency offluorescence energy transfer between fluorophores, and hence theirspatial proximity. Typically, measurements are performed at 0-37° C. asa function of time following the addition of the modifying enzyme (and,optionally, a modulator or candidate modulator of function for thatenzyme, as described below) to the system in 50 mM histidine pH 7.0, 120mM KCl, 5 mM MgSO₄, 5 mM NaF, 0.05 mM EGTA and 0.2 mM ATP. The assay maybe performed at a higher temperature if that temperature is compatiblewith the enzyme(s) under study.

[0206] Alternative Cell-Free Assay System of the Invention

[0207] A cell-free assay system according to the invention is requiredto permit binding of an unmodified, labeled natural binding domain,sequence or polypeptide and its binding partner to occur. As indicatedherein, such a system may comprise a low-ionic-strength buffer (e.g.,physiological salt, such as simple saline or phosphate- and/orTris-buffered saline or other as described above), a cell culturemedium, of which many are known in the art, or a whole or fractionatedcell lysate. The components of an assay of protein modificationaccording to the invention may be added into a buffer, medium or lysateor may have been expressed in cells from which a lysate is derived.Alternatively, a cell-free transcription- and/or translation system maybe used to deliver one or more of these components to the assay system.Nucleic acids of use in cell-free expression systems according to theinvention are as described for in vivo assays, below.

[0208] An assay of the invention may be peformed in a standard in vitrotranscription/translation system under conditions which permitexpression of a recombinant or other gene. The TNT® T7 Quick CoupledTranscription/Translation System (Cat. # L1170; Promega) contains allreagents necessary for in vitro transcription/translation except the DNAof interest and the detection label; as discussed below, polypeptidescomprising natural binding domains, sequences or polypeptides or theirbinding partners may be encoded by expression constructs in which theircoding sequences are fused in-frame to those encoding fluorescentproteins. The TNT® Coupled Reticulocyte Lysate Systems (comprising arabbit reticulocyte lysate) include: TNT® T3 Coupled Reticulocyte LysateSystem (Cat. # L4950; Promega); TNT® T7 Coupled Reticulocyte LysateSystem (Cat. # L4610; Promega); TNT® SP6 Coupled Reticulocyte LysateSystem (Cat. # L4600; Promega); TNT® T7/SP6 Coupled Reticulocyte LysateSystem (Cat. # L5020; Promega); TNT® T7/T3 Coupled Reticulocyte LysateSystem (Cat. # L5010; Promega).

[0209] An assay involving a cell lysate or a whole cell (see below) maybe performed in a cell lysate or whole cell preferably eukaryotic innature (such as yeast, fungi, insect, e.g., Drosophila), mouse, orhuman). An assay in which a cell lysate is used is performed in astandard in vitro system under conditions which permit gene expression.A rabbit reticulocyte lysate alone is also available from Promega,either nuclease-treated (Cat. # L4960) or untreated (Cat. # L4151).

[0210] Candidate Modulators of Protein Kinases and/or Phosphatases to beScreened According to the Invention

[0211] Whether in vitro or in an in vivo system (see below), theinvention encompasses methods by which to screen compositions which mayenhance, inhibit or not affect (e.g., in a cross-screening procedure inwhich the goal is to determine whether an agent intended for one purposeadditionally affects general cellular functions, of which proteinphosphorylation/dephosphorylation is an example) the activity of aprotein kinase or phosphatase.

[0212] Candidate modulator compounds from large libraries of syntheticor natural compounds can be screened. Numerous means are currently usedfor random and directed synthesis of saccharide, peptide, and nucleicacid based compounds. Synthetic compound libraries are commerciallyavailable from a number of companies including Maybridge Chemical Co.(Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), BrandonAssociates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Arare chemical library is available from Aldrich (Milwaukee, Wis.).Combinatorial libraries are available and can be prepared.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available from e.g., PanLaboratories (Bothell, Wash.) or MycoSearch (NC), or are readilyproduceable by methods well known in the art. Additionally, natural andsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical, and biochemical means.

[0213] Useful compounds may be found within numerous chemical classes,though typically they are organic compounds, including small organiccompounds. Small organic compounds have a molecular weight of more than50 yet less than about 2,500 daltons, preferably less than about 750,more preferably less than about 350 daltons. Exemplary classes includeheterocycles, peptides, saccharides, steroids, and the like. Thecompounds may be modified to enhance efficacy, stability, pharmaceuticalcompatibility, and the like. Structural identification of an agent maybe used to identify, generate, or screen additional agents. For example,where peptide agents are identified, they may be modified in a varietyof ways to enhance their stability, such as using an unnatural aminoacid, such as a D-amino acid, particularly D-alanine, by functionalizingthe amino or carboxylic terminus, e.g. for the amino group, acylation oralkylation, and for the carboxyl group, esterification or amidification,or the like.

[0214] Candidate modulators which may be screened according to themethods of the invention include receptors, enzymes, ligands, regulatoryfactors, and structural proteins. Candidate modulators also includenuclear proteins, cytoplasmic proteins, mitochondrial proteins, secretedproteins, plasmalemma-associated proteins, serum proteins, viralantigens, bacterial antigens, protozoal antigens and parasitic antigens.Candidate modulators additionally comprise proteins, lipoproteins,glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such asribozymes or antisense nucleic acids). Proteins or polypeptides whichcan be screened using the methods of the present invention includehormones, growth factors, neurotransmitters, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, structural proteins, viral antigens, parasitic antigens,bacterial antigens and antibodies (see below).

[0215] Candidate modulators which may be screened according to theinvention also include substances for which a test cell or organismmight be deficient or that might be clinically effective inhigher-than-normal concentration as well as those that are designed toeliminate the translation of unwanted proteins. Nucleic acids of useaccording to the invention not only may encode the candidate modulatorsdescribed above, but may eliminate or encode products which eliminatedeleterious proteins. Such nucleic acid sequences are antisense RNA andribozymes, as well as DNA expression constructs that encode them. Notethat antisense RNA molecules, ribozymes or genes encoding them may beadministered to a test cell or organism by a method of nucleic aciddelivery that is known in the art, as described below. Inactivatingnucleic acid sequences may encode a ribozyme or antisense RNA specificfor the a target MRNA. Ribozymes of the hammerhead class are thesmallest known, and lend themselves both to in vitro production anddelivery to cells (summarized by Sullivan, 1994, J. Invest. Dermatol.,103: 85S-98S; Usman et al., 1996, Curr. Opin. Struct. Biol., 6:527-533).

[0216] As stated above, antibodies are of use in the invention asmodulators (specifically, as inhibitors) of protein kinases and/orphosphatases. Methods for the preparation of antibodies are well knownin the art, and are briefly summarized as follows:

[0217] Either recombinant proteins or those derived from natural sourcescan be used to generate antibodies using standard techniques, well knownto those in the field. For example, the proteins are administered tochallenge a mammal such as a monkey, goat, rabbit or mouse. Theresulting antibodies can be collected as polyclonal sera, orantibody-producing cells from the challenged animal can be immortalized(e.g. by fusion with an immortalizing fusion partner) to producemonoclonal antibodies.

[0218] 1. Polyclonal Antibodies.

[0219] The antigen protein may be conjugated to a conventional carrierin order to increases its immunogenicity, and an antiserum to thepeptide-carrier conjugate is raised. Coupling of a peptide to a carrierprotein and immunizations may be performed as described (Dymecki et al.,1992, J. Biol. Chem., 267: 4815-4823). The serum is titered againstprotein antigen by ELISA (below) or alternatively by dot or spotblotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317).At the same time, the antiserum may be used in tissue sections preparedas described below. The serum is shown to react strongly with theappropriate peptides by ELISA, for example, following the procedures ofGreen et al., 1982, Cell, 28: 477-487.

[0220] 2. Monoclonal Antibodies.

[0221] Techniques for preparing monoclonal antibodies are well known,and monoclonal antibodies may be prepared using a candidate antigenwhose level is to be measured or which is to be either inactivated oraffinity-purified, preferably bound to a carrier, as described byArnheiter et al., Nature, 294, 278-280 (1981).

[0222] Monoclonal antibodies are typically obtained from hybridomatissue cultures or from ascites fluid obtained from animals into whichthe hybridoma tissue is introduced. Nevertheless, monoclonal antibodiesmay be described as being “raised to” or “induced by” a protein.

[0223] Monoclonal antibody-producing hybridomas (or polyclonal sera) canbe screened for antibody binding to the target protein. By antibody, weinclude constructions using the binding (variable) region of such anantibody, and other antibody modifications. Thus, an antibody useful inthe invention may comprise a whole antibody, an antibody fragment, apolyfunctional antibody aggregate, or in general a substance comprisingone or more specific binding sites from an antibody. The antibodyfragment may be a fragment such as an Fv, Fab or F(ab′)₂ fragment or aderivative thereof, such as a single chain Fv fragment. The antibody orantibody fragment may be non-recombinant, recombinant or humanized. Theantibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and soforth. In addition, an aggregate, polymer, derivative and conjugate ofan immunoglobulin or a fragment thereof can be used where appropriate.

[0224] Determination of Activity of Candidate Modulator of a ProteinKinase or Phosphatase

[0225] A candidate modulator of the activity of a protein kinase orphosphatase may be assayed according to the invention as describedherein, is determined to be effective if its use results in a differenceof about 10% or greater relative to controls in which it is not present(see below) in FRET resulting from the association of a labeled naturalbinding domain, sequence or polypeptide and its binding partner in thepresence of a protein-modifying enzyme.

[0226] The level of activity of a candidate modulator may be quantifiedusing any acceptable limits, for example, via the following formula:${{Percent}\quad {Modulation}} = {\frac{( {{Index}_{Control} - {Index}_{Sample}} )}{( {Index}_{Control} )} \times 100}$

[0227] where Index_(control) is the quantitative result (e.g., amountof- or rate of change in fluorescence at a given frequency, rate ofmolecular rotation, FRET, rate of change in FRET or other index ofmodification, including, but not limited to, enzyme inhibition oractivation) obtained in assays that lack the candidate modulator (inother words, untreated controls), and Index_(sample) represents theresult of the same measurement in assays containing the candidatemodulator. As described below, control measurements are made withdifferentially labeled natural binding domains, sequences orpolypeptides and their binding partners only, and then with thesemolecules plus a protein kinase or phosphatase which recognizes aphosphorylation site present on them.

[0228] Such a calculation is used in either in vitro or in vivo assaysperformed according to the invention.

[0229] B. In Vivo Assays of Enzymatic Activity According to theInvention

[0230] Reporter Group Protein Modification in Living Cells

[0231] Differentially-labeled natural binding domains, sequences orpolypeptides and their corresponding binding partners of the inventionare delivered (e.g., by microinjection) to cells, such as smooth musclecells (DDT1) or ventricular cardiac myocytes as previously described(Riabowol et al., 1988, Cold Spring Harbor Symposia on QuantitativeBiology, 53: 85-90). The ratio of emission from the labeled molecule(s)is measured as described above via a photomultiplier tube focused on asingle cell. Activation of a kinase (e.g., PKA by the addition ofdibutyryl cAMP or β-adrenergic agonists) is performed, subsequentinhibition is performed by removal of stimulus and by addition of asuitable antagonist (e.g., cAMP antagonist Rp-cAMPS).

[0232] Heterologous Expression of Peptides

[0233] Natural binding domains, sequences or polypeptides and/or theirbinding partners can be produced from the heterologous expression of DNAsequences that encode them or by chemical synthesis of the same.Expression can be in procaryotic or eukaryotic cells using a variety ofplasmid vectors capable of instructing heterologous expression.Purification of these products is achieved by destruction of the cells(e.g. French Press) and chromatographic purification of the products.This latter procedure can be simplified by the inclusion of an affinitypurification tag at one extreme of the peptide, separated from thepeptide by a protease cleavage site if necessary.

[0234] The Use of Cells or Whole Organisms in Assays of the Invention

[0235] When performed using cells, the assays of the invention arebroadly applicable to a host cell susceptible to transfection ortransformation including, but not limited to, bacteria (bothgram-positive and gram-negative), cultured- or explanted plant(including, but not limited to, tobacco, arabidopsis, carnation, riceand lentil cells or protoplasts), insect (e.g., cultured Drosophila ormoth cell lines) or vertebrate cells (e.g., mammalian cells) and yeast.

[0236] Organisms are currently being developed for the expression ofagents including DNA, RNA, proteins, non-proteinaceous compounds, andviruses. Such vector microorganisms include bacteria such as Clostridium(Parker et al., 1947, Proc. Soc. Exp. Biol. Med., 66: 461-465; Fox etal., 1996, Gene Therapy, 3: 173-178; Minton et al., 1995, FEMSMicrobiol. Rev., 17: 357-364), Salmonella (Pawelek et al., 1997, CancerRes., 57: 4537-4544; Saltzman et al., 1996, Cancer Biother. Radiopharm.,11: 145-153; Carrier et al., 1992, J. immunol., 148: 1176-1181; Su etal., 1992, Microbiol. Pathol., 13: 465-476; Chabalgoity et al., 1996,Infect. Immunol., 65: 2402-2412), Listeria (Schafer et al., 1992, J.Immunol., 149: 53-59; Pan et al., 1995, Nature Med., 1: 471-477) andShigella (Sizemore et al., 1995, Science, 270: 299-302), as well asyeast, mycobacteria, slime molds (members of the taxaDictyosteliida—such as of the genera Polysphondylium and Dictystelium,e.g. Dictyostelium discoideum—and Myxomycetes—e.g. of the generaPhysarum and Didymium) and members of the Domain Arachaea (including,but not limited to, archaebacteria), which have begun to be used inrecombinant nucleic acid work, members of the phylum Protista, or othercell of the algae, fungi, or any cell of the animal or plant kingdoms.

[0237] Plant cells useful in expressing polypeptides of use in assays ofthe invention include, but are not limited to, tobacco (Nicotianaplumbaginifolia and Nicotiana tabacum), arabidopsis (Arabidopsisthaliana), Aspergillus niger, Brassica napus, Brassica nigra, Daturainnoxia, Vicia narbonensis, Vicia faba, pea (Pisum sativum),cauliflower, carnation and lentil (Lens culinaris). Either whole plants,cells or protoplasts may be transfected with a nucleic acid of choice.Methods for plant cell transfection or stable transformation includeinoculation with Agrobacterium tumefaciens cells carrying the constructof interest (see, among others, Turpen et al., 1993, J. Virol. Methods,42: 227-239), administration of liposome-associated nucleic acidmolecules (Maccarrone et al., 1992, Biochem. Biophys. Res. Commun., 186:1417-1422) and microparticle injection (Johnston and Tang, 1993, Genet.Eng. (NY), 15: 225-236), among other methods. A generally useful planttranscriptional control element is the cauliflower mosaic virus (CaMV)35S promoter (see, for example, Saalbach et al., 1994, Mol. Gen. Genet.,242: 226-236). Non-limiting examples of nucleic acid vectors useful inplants include pGSGLUC1 (Saalbach et al., 1994, supra), pGA492 (Perez etal., 1989, Plant Mol. Biol., 13: 365-373), pOCA18 (Olszewski et al.,1988, Nucleic Acids Res., 16: 10765-10782), the Ti plasmid (Roussell etal., 1988, Mol. Gen. Genet., 211: 202-209) and pKR612B1 (Balazs et al.,1985, Gene, 40: 343-348).

[0238] Mammalian cells are of use in the invention. Such cells include,but are not limited to, neuronal cells (those of both primary explantsand of established cell culture lines) cells of the immune system (suchas T-cells, B-cells and macrophages), fibroblasts, hematopoietic cellsand dendritic cells. Using established technologies, stem cells (e.g.hematopoietic stem cells) may be used for gene transfer after enrichmentprocedures. Alternatively, unseparated hematopoietic cells and stem cellpopulations may be made susceptible to DNA uptake. Transfection ofhematopoietic stem cells is described in Mannion-Henderson et al., 1995,Exp. Hematol., 23: 1628; Schiffmann et al., 1995, Blood, 86: 1218;Williams, 1990, Bone Marrow Transplant, 5: 141; Boggs, 1990, Int. J.Cell Cloning, 8: 80; Martensson et al., 1987, Eur. J. Immunol., 17:1499; Okabe et al., 1992, Eur. J. Immunol., 22: 37-43; and Banerji etal., 1983, Cell, 33: 729. Such methods may advantageously be usedaccording to the present invention.

[0239] Nucleic Acid Vectors for the Expression of Assay Components ofthe Invention in Cells or Multicellular Organisms

[0240] A nucleic acid of use according to the methods of the inventionmay be either double- or single stranded and either naked or associatedwith protein, carbohydrate, proteoglycan and/or lipid or othermolecules. Such vectors may contain modified and/or unmodifiednucleotides or ribonucleotides. In the event that the gene to betransfected may be without its native transcriptional regulatorysequences, the vector must provide such sequences to the gene, so thatit can be expressed once inside the target cell. Such sequences maydirect transcription in a tissue-specific manner, thereby limitingexpression of the gene to its target cell population, even if it istaken up by other surrounding cells. Alternatively, such sequences maybe general regulators of transcription, such as those that regulatehousekeeping genes, which will allow for expression of the transfectedgene in more than one cell type; this assumes that the majority ofvector molecules will associate preferentially with the cells of thetissue into which they were injected, and that leakage of the vectorinto other cell types will not be significantly deleterious to therecipient organism. It is also possible to design a vector that willexpress the gene of choice in the target cells at a specific time, byusing an inducible promoter, which will not direct transcription unlessa specific stimulus, such as heat shock, is applied.

[0241] A gene encoding a component of the assay system of the inventionor a candidate modulator of protein kinase or phosphatase activity maybe transfected into a cell or organism using a viral or non-viral DNA orRNA vector, where non-viral vectors include, but are not limited to,plasmids, linear nucleic acid molecules, artificial chromomosomes andepisomal vectors. Expression of heterologous genes in mammals has beenobserved after injection of plasmid DNA into muscle (Wolff J. A. et al.,1990, Science, 247: 1465-1468; Carson D. A. et al., U.S. Pat. No.5,580,859), thyroid (Sykes et al., 1994, Human Gene Ther., 5: 837-844),melanoma (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge etal., 1995, Nature Genet., 10: 161-166), liver (Hickman et al., 1994,Human Gene Therapy, 5: 1477-1483) and after exposure of airwayepithelium (Meyer et al., 1995, Gene Therapy, 2: 450-460).

[0242] In addition to vectors of the broad classes described above andfusion gene expression construct encoding a natural binding domain,sequence or polypeptide fused in-frame to a fluorescent protein, asdescribed above (see “Fluorescent resonance energy transfer”), microbialplasmids, such as those of bacteria and yeast, are of use in theinvention.

[0243] Bacterial plasmids:

[0244] Of the frequently used origins of replication, pBR322 is usefulaccording to the invention, and pUC is preferred. Although notpreferred, other plasmids which are useful according to the inventionare those which require the presence of plasmid encoded proteins forreplication, for example, those comprising pT181, FII, and FI origins ofreplication.

[0245] Examples of origins of replication which are useful in assays ofthe invention in E. coli and S. typhimurium include but are not limitedto, pHETK (Garapin et al., 1981, Proc. Natl. Acad. Sci. U.S.A., 78:815-819), p279 (Talmadge et al., 1980, Proc. Natl. Acad. Sci. U.S.A.,77: 3369-3373), p5-3 and p21A-2 (both from Pawalek et al., 1997, CancerRes., 57: 4537-4544), pMB1 (Bolivar et al., 1977, Gene, 2: 95-113),ColE1 (Kahn et al., 1979, Methods Enzymol., 68: 268-280), p15A (Chang etal., 1978, J. Bacteriol., 134: 1141-1156); pSC101 (Stoker et al., 1982,Gene, 18: 335-341); R6K (Kahn et al., 1979, supra); R1 (temperaturedependent origin of replication, Uhlin et al., 1983, Gene, 22: 255-265);lambda dv (Jackson et al., 1972, Proc. Nat. Aca. Sci. U.S.A., 69:2904-2909); pYA (Nakayama et al., 1988, infra). An example of an originof replication that is useful in Staphylococcus is pT181 (Scott, 1984,Microbial Reviews 48: 1-23). Of the above-described origins ofreplication, pMB1, p15A and ColE1 are preferred because these origins donot require plasmid-encoded proteins for replication.

[0246] Yeast plasmids:

[0247] Three systems are used for recombinant plasmid expression andreplication in yeasts:

[0248] 1. Integrating. An example of such a plasmid is YIp, which ismaintained at one copy per haploid genome, and is inherited in Mendelianfashion. Such a plasmid, containing a gene of interest, a bacterialorigin of replication and a selectable gene (typically anantibiotic-resistance marker), is produced in bacteria. The purifiedvector is linearized within the selectable gene and used to transformcompetent yeast cells. Regardless of the type of plasmid used, yeastcells are typically transformed by chemical methods (e.g. as describedby Rose et al., 1990, Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The cells are treated withlithium acetate to achieve transformation efficiencies of approximately10⁴ colony-forming units (transformed cells)/μg of DNA. Yeast performhomologous recombination such that the cut, selectable marker recombineswith the mutated (usually a point mutation or a small deletion) hostgene to restore function. Transformed cells are then isolated onselective media.

[0249] 2. Low copy-number ARS-CEN, of which YCp is an example. Such aplasmid contains the autonomous replicating sequence (ARS1), a sequenceof approximately 700 bp which, when carried on a plasmid, permits itsreplication in yeast, and a centromeric sequence (CEN4), the latter ofwhich allows mitotic stability. These are usually present at 1-2 copiesper cell. Removal of the CEN sequence yields a YRp plasmid, which istypically present in 100-200 copes per cell; however, this plasmid isboth mitotically and meiotically unstable.

[0250] 3. High-copy-number 2μ circles. These plasmids contain a sequenceapproximately 1 kb in length, the 2μ sequence, which acts as a yeastreplicon giving rise to higher plasmid copy number; however, theseplasmids are unstable and require selection for maintenance. Copy numberis increased by having on the plasmid a selection gene operativelylinked to a crippled promoter. This is usually the LEU2 gene with atruncated promoter (LEU2-d), such that low levels of the Leu2p proteinare produced; therefore, selection on a leucine-depleted medium forcesan increase in copy number in order to make an amount of Leu2psufficient for cell growth.

[0251] As suggested above, examples of yeast plasmids useful in theinvention include the YRp plasmids (based on autonomously-replicatingsequences, or ARS) and the YEp plasmids (based on the 2μ circle), ofwhich examples are YEp24 and the YEplac series of plasmids (Gietz andSugino, 1988, Gene, 74: 527-534). (See Sikorski, “Extrachromosomalcloning vectors of Saccharomyces cerevisiae”, in Plasmids, A PracticalApproach, Ed. K. G. Hardy, IRL Press, 1993; and Yeast Cloning Vectorsand Genes, Current Protocols in Molecular Biology, Section II, Unit13.4, Eds., Ausubel et al., 1994).

[0252] In addition to a yeast origin of replication, yeast plasmidsequences typically comprise an antibiotic resistance gene, a bacterialorigin of replication (for propagation in bacterial cells) and a yeastnutritional gene for maintenance in yeast cells. The nutritional gene(or “auxotrophic marker”) is most often one of the following (with thegene product listed in parentheses and the sizes quoted encompassing thecoding sequence, together with the promoter and terminator elementsrequired for correct expression):

[0253] TRP1 (PhosphoADP-ribosylanthranilate isomerase, which is acomponent of the tryptophan biosynthetic pathway).

[0254] URA3 (Orotidine-5′-phosphate decarboxylase, which takes part inthe uracil biosynthetic pathway).

[0255] LEU2 (3-Isopropylmalate dehydrogenase, which is involved with theleucine biosynthetic pathway).

[0256] HIS3 (Imidazoleglycerolphosphate dehydratase, or IGPdehydratase).

[0257] LYS2 (α-aminoadipate-semialdehyde dehydrogenase, part of thelysine biosynthetic pathway).

[0258] Alternatively, the screening system may operate in an intact,living multicellular organism, such as an insect or a mammal. Methods ofgenerating transgenic Drosophila, mice and other organisms, bothtransiently and stably, are well known in the art; detection offluorescence resulting from the expression of Green Fluorescent Proteinin live Drosophila is well known in the art. One or more gene expressionconstructs encoding one or more of a labeled natural binding domain,sequence or polypeptide, a binding partner, a protein kinase orphosphatase and, optionally, a candidate modulator thereof areintroduced into the test organism by methods well known in the art (seealso below). Sufficient time is allowed to pass after administration ofthe nucleic acid molecule to allow for gene expression, for binding of anatural binding domain, sequence or polypeptide to its binding partnerand for chromophore maturation, if necessary (e.g., Green FluorescentProtein matures over a period of approximately 2 hours prior tofluorescence) before FRET is measured. A reaction component(particularly a candidate modulator of enzyme function) which is notadministered as a nucleic acid molecule may be delivered by a methodselected from those described below.

[0259] Dosage and Administration of a Labeled Natural Binding DomainSequence or Polypeptide, Binding Partner Therefor, Protein Kinase orPhosphatase or Candidate Modulator thereof for Use in an In Vivo Assayof the Invention

[0260] Dosage

[0261] For example, the amount of each labeled natural binding domain orbinding partner therefor must fall within the detection limits of thefluorescence-measuring device employed. The amount of an enzmye orcandidate modulator thereof will typically be in the range of about 1μg-100 mg/kg body weight. Where the candidate modulator is a peptide orpolypeptide, it is typically administered in the range of about 100-500μg/ml per dose. A single dose of a candidate modulator, or multipledoses of such a substance, daily, weekly, or intermittently, iscontemplated according to the invention.

[0262] A candidate modulator is tested in a concentration range thatdepends upon the molecular weight of the molecule and the type of assay.For example, for inhibition of protein/protein or protein/DNA complexformation or transcription initiation (depending upon the level at whichthe candidate modulator is thought or intended to modulate the activityof a protein kinase or phosphatase according to the invention), smallmolecules (as defined above) may be tested in a concentration range of 1pg-100 μg/ml, preferably at about 100 pg-10 ng/ml; large molecules,e.g., peptides, may be tested in the range of 10 ng-100 μg/ml,preferably 100 ng-10 μg/ml.

[0263] Administration

[0264] Generally, nucleic acid molecules are administered in a mannercompatible with the dosage formulation, and in such amount as will beeffective. In the case of a recombinant nucleic acid encoding a naturalbinding domain and/or binding partner therefor, such an amount should besufficient to result in production of a detectable amount of the labeledprotein or peptide, as discussed above. In the case of a protein kinaseor phosphatase, the amount produced by expression of a nucleic acidmolecule should be sufficient to ensure that at least 10% of naturalbinding domains or binding partners therefor will undergo modificationif they comprise a target site recognized by the enzyme being assayed.Lastly, the amount of a nucleic acid encoding a candidate modulator of aprotein kinase or phosphatase of the invention must be sufficient toensure production of an amount of the candidate modulator which can, ifeffective, produce a change of at least 10% in the effect of the targetprotein kinase or phosphatase on FRET or other label emission resultingfrom binding of a natural binding domain to its binding partner or, ifadministered to a patient, an amount which is prophylactically and/ortherapeutically effective.

[0265] When the end product (e.g. an antisense RNA molecule or ribozyme)is administered directly, the dosage to be administered is directlyproportional to the amount needed per cell and the number of cells to betransfected, with a correction factor for the efficiency of uptake ofthe molecules. In cases in which a gene must be expressed from thenucleic acid molecules, the strength of the associated transcriptionalregulatory sequences also must be considered in calculating the numberof nucleic acid molecules per target cell that will result in adequatelevels of the encoded product. Suitable dosage ranges are on the orderof, where a gene expression construct is administered, 0.5-to 1 μg, or1-10 μg, or optionally 10-100 μg of nucleic acid in a single dose. It isconceivable that dosages of up to 1 mg may be advantageously used. Notethat the number of molar equivalents per cell vary with the size of theconstruct, and that absolute amounts of DNA used should be adjustedaccordingly to ensure adequate gene copy number when large constructsare injected.

[0266] If no effect (e.g., of a protein kinase or phosphatase or aninhibitor thereof) is seen within four orders of magnitude in eitherdirection of the starting dosage, it is likely that a protein kinase orphosphatase does not recognize the target site of the natural bindingdomain (and, optionally, its binding partner) according to theinvention, or that the candidate modulator thereof is not of useaccording to the invention. It is critical to note that when highdosages are used, the concentration must be kept below harmful levels,which may be known if an enzyme or candidate modulator is a drug that isapproved for clinical use. Such a dosage should be one (or, preferably,two or more) orders of magnitude below the LD₅₀ value that is known fora laboratory mammal, and preferably below concentrations that aredocumented as producing serious, if non-lethal, side effects.

[0267] Components of screening assays of the invention may be formulatedin a physiologically acceptable diluent such as water, phosphatebuffered saline, or saline, and further may include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are materials well known in the art.Administration of labeled polypeptides comprising a natural bindingdomain, sequence, polypeptide or a binding partner therefor, a proteinkinase or phosphatase or a candidate modulator as described herein maybe either localized or systemic.

[0268] Localized administration:

[0269] Localized administration of a component of an assay of theinvention is preferably by via injection or by means of a drip device,drug pump or drug-saturated solid matrix from which the labeled naturalbinding domain, sequence or polypeptide, binding partner therefor,protein kinase or phosphatase or candidate modulator therefor, ornucleic acid encoding any of these can diffuse implanted at the targetsite. When a tissue that is the target of delivery according to theinvention is on a surface of an organism, topical administration of apharmaceutical composition is possible.

[0270] Compositions comprising a composition of- or of use in theinvention which are suitable for topical administration can take one ofseveral physical forms, as summarized below:

[0271] (i) A liquid, such as a tincture or lotion, which may be appliedby pouring, dropping or “painting” (i.e. spreading manually or with abrush or other applicator such as a spatula) or injection.

[0272] (ii) An ointment or cream, which may be spread either manually orwith a brush or other applicator (e.g. a spatula), or may be extrudedthrough a nozzle or other small opening from a container such as acollapsible tube.

[0273] (iii) A dry powder, which may be shaken or sifted onto the targettissue or, alternatively, applied as a nebulized spray.

[0274] (iv) A liquid-based aerosol, which may be dispensed from acontainer selected from the group that comprises pressure-driven spraybottles (such as are activated by squeezing), natural atomizers (or“pump-spray” bottles that work without a compressed propellant) orpressurized canisters.

[0275] (v) A carbowax or glycerin preparation, such as a suppository,which may be used for rectal or vaginal administration of a therapeuticcomposition.

[0276] In a specialized instance, the tissue to which a candidatemodulator of a protein kinase or phosphatase is to be delivered forassay (or, if found effective, for therapeutic use) is the lung. In sucha case the route of administration is via inhalation, either of a liquidaerosol or of a nebulized powder of. Drug delivery by inhalation,whether for topical or systemic distribution, is well known in the artfor the treatment of asthma, bronchitis and anaphylaxis. In particular,it has been demonstrated that it is possible to deliver a protein viaaerosol inhalation such that it retains its native activity in vivo (seeHubbard et al., 1989, J. Clin. Invest., 84: 1349-1354).

[0277] Systemic administration:

[0278] Systemic administration of a protein, nucleic acid or other agentaccording to the invention may be performed by methods of whole-bodydrug delivery are well known in the art. These include, but are notlimited to, intravenous drip or injection, subcutaneous, intramuscular,intraperitoneal, intracranial and spinal injection, ingestion via theoral route, inhalation, trans-epithelial diffusion (such as via adrug-impregnated, adhesive patch) or by the use of an implantable,time-release drug delivery device, which may comprise a reservoir ofexogenously-produced protein, nucleic acid or other material or may,instead, comprise cells that produce and secrete a natural bindingdomain and/or a binding partner therefor, protein kinase or phosphataseor candidate modulator thereof. Note that injection may be performedeither by conventional means (i.e. using a hypodermic needle) or byhypospray (see Clarke and Woodland, 1975, Rheumatol. Rehabil., 14:47-49). Components of assays of the invention can be given in a single-or multiple dose.

[0279] Delivery of a nucleic acid may be performed using a deliverytechnique selected from the group that includes, but is not limited to,the use of viral vectors and non-viral vectors, such as episomalvectors, artificial chromosomes, liposomes, cationic peptides,tissue-specific cell transfection and transplantation, administration ofgenes in general vectors with tissue-specific promoters, etc.

[0280] Kits According to the Invention

[0281] A Kit for Assaying the Activity of a Protein Kinase orPhosphatase

[0282] In order to facilitate convenient and widespread use of theinvention, a kit is provided which contains the essential components forscreening the activity of a protein kinase or phosphatase, as describedabove. A natural binding domain, sequence or polypeptide, as definedabove, and its corresponding binding partner are provided, as is asuitable reaction buffer for in vitro assay or, alternatively, cells ora cell lysate. A reaction buffer which is “suitable” is one which ispermissive of the activity of the enzyme to be assayed and which permitsphosphorylation-dependent binding of the natural binding domain to thebinding partner. The labeled polypeptide components are provided aspeptide/protein or a nucleic acid comprising a gene expression constructencoding the one or more of a peptide/protein, as discussed above.Natural binding domains, sequences and polypeptides, as well as theircorresponding binding partners, are supplied in a kit of the inventioneither in solution (preferably refrigerated or frozen) in a buffer whichinhibits degradation and maintains biological activity, or are providedin dried form, i.e., lyophilized. In the latter case, the components areresuspended prior to use in the reaction buffer or other biocompatiblesolution (e.g. water, containing one or more of physiological salts, aweak buffer, such as phosphate or Tris, and a stabilizing substance suchas glycerol, sucrose or polyethylene glycol); in the latter case, theresuspension buffer should not inhibit phosphorylation-dependent bindingof the natural binding domain, sequence or polypeptide with the bindingpartner when added to the reaction buffer in an amount necessary todeliver sufficient protein for an assay reaction. Natural bindingdomains, sequences or polypeptides or their binding partners provided asnucleic acids are supplied- or resuspended in a buffer which permitseither transfection/transformation into a cell or organism or in vitrotranscription/translation, as described above. Each of these componentsis supplied separately contained or in admixture with one or more of theothers in a container selected from the group that includes, but is notlimited to, a tube, vial, syringe or bottle.

[0283] Optionally, the kit includes cells. Eukaryotic or prokaryoticcells, as described above, are supplied in- or on a liquid or solidphysiological buffer or culture medium (e.g. in suspension, in a stabculture or on a culture plate, e.g. a Petri dish). For ease of shipping,the cells are typically refrigerated, frozen or lyophilized in a bottle,tube or vial. Methods of cell preservation are widely known in the art;suitable buffers and media are widely known in the art, and are obtainedfrom commerical suppliers (e.g., Gibco/LifeTechnologies) or made bystandard methods (see, for example Sambrook et al., 1989, MolecularCloning. A Laboratory Manual., 2nd Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

[0284] An enzyme being assayed according to the invention is added tothe assay system either as a protein (isolated, partially-purified orpresent in a crude preparation such as a cell extract or even a livingcell) or a recombinant nucleic acid. Methods of expressing a nucleicacid comprising an enzyme or other protein are well known in the art(see again above).

[0285] An assay of the invention is carried out using the kit accordingto the methods described above and in the Examples.

[0286] A Kit for Screening a Candidate Modulator of Protein Kinase orPhosphatase Activity According to the Invention

[0287] A candidate modulator of post-translational phosphorylation ordephosphorylation may be assayed using a kit of the invention. A kit asdescribed above is used for this application, with the assay performedfurther comprising the addition of a candidate modulator of the proteinkinase or phosphatase which is present to the reaction system.Optionally, a protein kinase or phosphatase is supplied with the kit,either as a protein or nucleic acid as described above.

[0288] Assays of protein activity are performed as described above. At aminimum, three detections are performed, one in which the naturalbinding domain and binding partner are present without the proteinkinase or phosphatase or candidate modulator thereof (control reactionA), one in which the polypeptides are incubated with the modifyingenzyme under conditions which permit the phosphorylation ordephosphorylation reaction to occur (control reaction B) and one inwhich the protein kinase or phosphatase and candidate modulator are bothincubated with the labeled polypeptides under conditions which permitthe modification reaction to occur (test reaction). In each case,conditions are suitable to permit phosphorylation-dependent associationof the natural binding domain, sequence or polypeptide and the bindingpartner. The result of the last detection procedure is compared withthose of the two controls; the candidate modulator is judged to beefficacious if there is a shift in either of the observed amount ofsignal (i.e., total amount- or rate of change of fluorescence, FRET,mass of a protein complex or inhibition or activation of an enzyme) ofat least 10% away from that observed in control reaction B toward thatobserved in control reaction A.

EXAMPLE 1 Use of a Polypeptide Comprising a Natural Binding Domain as aPhosphorylation Reporter According to the Invention: Assay 1

[0289] An assay of this type involves the following components:

[0290] v-Src SH2 domain (amino acids 148-246; Waksman et al., 1993,Cell, 72: 779-790; OWL database accession no. M33292), and

[0291] Hamster polyomavirus middle T antigen (Ag, below) (321-331,EPQYEEIPIYL; Waksman et al., 1993, supra; OWL database accession no.P03079).

[0292] SH2 domains are found in proteins involved in a number ofsignalling pathways and their binding to specific phosphorylatedtyrosine residues is key in mediating the transmission of signalsbetween tyrosine kinases and the proteins in the cell which respond totyrosine phosphorylation (Waksman et al., 1993, supra and referencestherein). Individual SH2 domains recognize specific sequences, and thesequence specificity of a number of SH2 domains has been determined(Songyang et al., 1993, supra) using a phosphopeptide library. Thesedata provide a number of possible domain/peptide pairs which are usefulin assays of enzymatic activity according to the invention. The crystalstructure of the Src SH2 domain complexed with a peptide containing itsspecific recognition motif from the hamster middle-T antigen (targettyrosine for phosphorylation shown in bold above) has been determined byWaksman et. al.(Cell 72, 779-790).

[0293] Thus, the assay is:

[0294] F1 is the donor fluorophore, F2 the acceptor fluorophore and Pdenotes the addition of a phosphate group to the target tyrosineresidue.

[0295] The peptide as used in the crystallization described above doesnot contain suitable residues for convenient labelling, and a labelwithin this short sequence is too close to the phosphorylation site. Ashort linker (e.g., Gly-Gly) is, therefore, added to either the C- orN-terminus of the peptide with a residue such as Lys for labelling onthe end. The location of this linker will depend upon the location of F1in the SH2 domain.

[0296] A number of potential locations for the fluorophore in the SH2domain have been identified based upon crystal structure: SH2 DomainMiddle T Ag. peptide K 232 C-terminal extension (G-G-K or similar) R 217C-terminal extension K 181 N-terminal extension R 156** N-terminalextension

[0297] If a fluorescent protein (e.g., Green Fluorescent Protein, GFP)is used instead of a chemical fluorophore, it is placed at the N-terminiof both the SH2 domain and the peptide

EXAMPLE 2 Use of a Polypeptide Comprising a Natural Binding Domain as aPhosphorylation Reporter According to the Invention: Assay 2

[0298] This assay involves the following components:

[0299] PTB domain of IRS-1 (amino acids 157-267) (Zhou et al., 1996,Nature Structural Biology, 3: 388-393; OWL accession no. P35568), and

[0300] Interleukin 4 Receptor (IL-4R) (amino acids 489-499, LVIAGNPAYRS;Zhou et. al., 1996, supra; OWL accession no. P24394)

[0301] Phosphotyrosine binding (PTB) domains are found in a number ofproteins involved in signalling pathways utilizing tyrosinephosphorylation. The PTB domain has functional similarities to the SH2domain but differs in its mechanism of action and structure, as well asin sequence recognition (Laminet et al., 1996, J. Biol. Chem., 271:264-269; Zhou et. al., 1996, supra and references therein). These twoclasses of domain have little sequence identity. NMR structural analysisof the PTB domain of IRS-1 complexed with the IL-4 receptor peptide hasbeen performed (Zhou et al., 1996, supra).

[0302] The assay format is as follows:

[0303] F1 is the donor fluorophore, F2 the acceptor fluorophore, and Pdenotes the addition of a phosphate group to the target tyrosineresidue.

[0304] The peptide of the NMR study described above does not containsuitable residues for convenient labelling except the arginine next tothe phosphorylation site, and a label within this short sequence may betoo close to the target site for phosphorylation. A short linker may beadded to either the C- or N-terminus of the peptide with a residue forlabelling on the end. The location of such a linker depends upon thelocation of F1 in the PTB domain.

[0305] Several potential locations for the fluorophore in the PTBsequence have been identified from the NMR structure: PTB domain IL-4Rpeptide K161 N-terminal extension (G-G-K or similar) K190 N-terminalextension N-terminal extension N-terminal extension C-terminal extensionC-terminal extension

[0306] Again, if GFP is used in lieu of a chemical fluorophore, it canbe fused in-frame to either the N- or C-terminus of both the PTBsequence and the binding partner.

EXAMPLE 3

[0307] An assay analogous to that in Example 2 can be configuredaccording to the invention using the PTB domain of the proto-oncogeneproduct Cb1 and a peptide derived from the Zap-70 tyrosine kinase. TheCb1 phosphotyrosine-binding domain selects a D(N/D)XpY motif and bindsto the Tyr₂₉₂ negative regulatory phosphorylation site of ZAP-70 (Lupheret al., 1997, J. Biol. Chem., 272: 33140-33144).

[0308] The components of the assay are:

[0309] The Cb1 N-terminal domain (amino acids 1-357; Lupher et al.,1996, J. Biol Chem., 271: 24063-24068; OWL accession no. P22681), and

[0310] Zap-70 (amino acids 284-299, NH₃-IDTLNSDGYTPEPARI-COOH; Lupheret. al., 1996, supra; OWL accession no. P43403)

EXAMPLE 4 Use of a Polypeptide Comprising a Natural Binding Domain as aPhosphorylation Reporter According to the Invention: Assay 4

[0311] This assay involves the following component—

[0312] c-Src (residues 86-536; Xu et al., 1997, Nature, 385: 595-602;GenBank Accession No. K03218).

[0313] As stated above, Src is a member of a family of non-receptortyrosine kinases involved in the regulation of responses toextracellular signals. Association of src with both the plasma membraneand intracellular membranes is mediated by myristoylation at theN-terminus. The enzyme has four regions which are conserved throughoutthe family, the SH2 domain, the SH3 domain, the kinase or SH1 domain andthe C-terminal tail. In addition there is a unique region which does nothave homology between family members (Brown and Cooper, 1996, Biochim.Biophys. Acta, 1287: 121-149).

[0314] The SH2 domain binds tightly to specific tyrosine phosphorylatedsequences. This affinity plays a role in the interaction between src andother cellular proteins and also in the regulation of the kinase byphosphorylation. The C-terminal tail of src can be phosphorylated onTyr₅₃₀, which phosphorylation leads to almost complete inhibition ofkinase activity. There is strong evidence that this inhibition isachieved by the interaction of the C-terminal tail with the SH2 domain.This interaction is thought to promote a conformational change to the‘closed’ conformation which is further stabilized by the participationof the SH3 and kinase domains in intramolecular contacts.

[0315] The assay is diagramed as follows:

[0316] where F1 is the donor fluorophore, F2 is the acceptor fluorophoreand P denotes the addition of a phosphate group to the target tyrosineresidue.

[0317] There are several potential sites for labelling in thisstructure. Some examples of target residues are shown below: C-Terminaltail SH2 domain E527 D195, K198 C-Terminal extension (eg. Gly-Gly-Lys)R220, K235,

[0318] When a fluorescent protein is used in an assay such as this,using an intramolecular interaction to follow chemical modification, itis appropriate to place GFP between domains using a flexible linker topreserve protein domain interactions. This allows the GFP variants toapproach more closely and increase the efficiency of the FRET achieved,but must be balanced by the need to achieve a good distance betweenvariants in the ‘No FRET’ state. If sufficient spacing between donor andacceptor fluorophores or, alternatively, between a fluorophore or otherlabel and a quencher therefor, is not achieved in this manner, othercandidate locations for fluorescent protein fusion include, but are notlimited to, the C-terminus and the region between the SH2 domain and theSH2-kinase linker.

EXAMPLE 5 Solution FRET Assay for Yersinia Tyrosine Phosphatase (YOP)Using a Natural Binding Partner Labelled with a Fluorescent Protein anda Synthetic Peptide Labelled with a Chemical Fluorophore

[0319] The following solution based assay was performed to detect YOPactivity by measuring disruption of a complex between a fluorescentlylabelled SH2 domain of ZAP-70 and a synthetic peptide based on the TCRζchain labelled with a chemical fluorophore.

[0320] A FRET partnership is formed between the SH2 domain of ZAP-70 anda phosphorylated peptide based on the TCRζ chain, providing bothpartners are labelled with suitable fluorophores. Formation of FRET isfollowed in real-time by adding the two binding partners together.Disruption of FRET can be achieved by the addition of a phosphatase,which removes the phosphate required for the interaction of thepartners.

[0321] Methods:

[0322] TCR Peptide Sequence

[0323] Peptide 1. Phosphorylated TCRζ chain:

[0324] RCKFSRSAEPPAYQQGQNQLY_((p))NELNLGRREEY_((p))DVLD

[0325] Peptide Labelling

[0326] Peptide 1 was labelled with rhodamine under mild conditions usingthiol directed chemistry. 230 μM peptide was labelled in 20 mM TES pH 7in the presence of a three-fold excess of rhodamine-6-maleimide(Molecular Probes). Dialysis was utilised to remove excess dye from thepeptide. Labelling was verified by MALDI-TOF MS.

[0327] ZAP-GFP Cloning and Purification

[0328] DNA Constructs

[0329] ZAP-GFP: Primers were designed based on the published ZAP-70 DNAsequence (Genbank accession number L05148). The SH2 domain (amino acids1-259) of ZAP70 was cloned by PCR using the following oligo-nucleotides:Forward primer GGGATCCATATGCCAGACCCCGCGGCGCACCTG Reverse PrimerGGAATTCGGGCACTGCTGTTGGGGCAGGCCTCC

[0330] The resultant PCR fragment was digested with BamHI and EcoRI andinserted into pET28a (Novagen) to generate vector pFS45. DNA encodingGFP in the vector pQBI25-FNI (Quantum) was digested with MluI and theresultant 5′ overhang was “filled in” using T4 DNA polymerase (NEB).After the polymerase was denatured by heat treatment the DNA was furtherdigested with EcoRI and the resultant 850 bp band was gel purified. Thevector pFS45 was digested with HindIII and the resultant 5′ overhang was“filled in” with T4 DNA polymerase and then further digested with EcoRI.After the digested vector was gel purified it was ligated with thepurified DNA encoding GFP to generate pFS46 which was designed toexpress a ZAP70-GFP fusion protein in bacteria.

[0331] Expression and Purification Procedure

[0332] Fresh transformants of ZAP-GFP pET-28a in BRL(DE3) were used toinoculate 3 ml LB/kanamycin (100 μg/ml). The starter cultures wereincubated overnight at 37° C. with constant shaking. From these startercultures 1 ml was used to inoculate 400 ml Terrific Broth/kanamycin (100μg/ml) in a 2L, baffled flask. Cultures were incubated at 37° C. at 200rpm for approximately 5 hrs until the OD600 nm reached 0.5 Abs units. Atthis point cultures were induced by adding IPTG to a concentration of 1mM. The cultures were then left incubating at room temperature overnightwith gentle shaking on a benchtop rotator. Bacteria were harvested bycentrifugation at 3000 rpm for 20 mins. The bacterial pellet wasresuspended in 25 ml lysis buffer (50 mM phosphate buffer pH 7.0, 300 mMNaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75 mg/ml Lysozyme).Lysis of the resuspended cells was initiated by gentle stirring for 1 hrat room temperature. The partially lysed mixture was subjected to 2cycles of freeze thawing in liquid nitrogen. Finally the cells weresonicated on ice using a 10 mm probe at high power. Sonication wasperformed on a pulse setting for a period of 3 min. The crude lysate wasthen centrifuged at 15,000. rpm for 30 mins to remove cell debris.Hexa-His tagged proteins were purified from the clear lysate usingTALON® resin (Clontech). Proteins were bound to the resin in a batchwisemanner by gentle shaking at room temperature for 30 min. Non-His taggedproteins were removed by washing the resin at least twice with 10× bedvolume of wash buffer (50 mM sodium phosphate pH 7.0, 30 mM NaCl, 5 mMfluorescence-blank imidazole ). The washed resin was loaded into a 2 mlcolumn and the bound proteins were released with elution buffer (50 mMsodium phosphate pH 7.0, 300 mM NaCl, 150 mM fluorescence-blankImidazole). Elution was normally achieved after the first 0.5 ml andwithin 2-3 ml in total. Proteins were stored at −80° C. after snapfreezing in liquid nitrogen in the presence of 10% glycerol.

[0333] Formation of the FRET Partnership

[0334] ZAP-GFP was diluted to 0.5 μM in YOP assay buffer (50 mM Tris-HClpH 7.2, 10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35). 98 μlof this solution was used per assay. Initial readings of thefluorescence of the ZAP-GFP construct were made using 485 nm excitationwavelength and 520 nm emission wavelength. Rhodamine labelled peptide (2μl of a 115 μM peptide solution) was added to the ZAP-GFP and formationof FRET followed in real-time by measuring the decrease in fluorescenceemission of ZAP-GFP at 520 nm.

[0335] YOP Source and Assay Conditions

[0336] Yersinia protein tyrosine phosphatase (YOP) was purchased fromUpstate Biotechnology. YOP, 3 units, was added to the ZAP-GFP/peptideFRET mixture and the increase in fluorescence emission at 520 nm, wasfollowed in real-time as the partnership was disrupted (FIG. 4).Dependence of YOP activity on TCR peptide concentration and Kmdetermination was measured using different concentrations of rhodaminelabelled TCRζ peptide (FIG. 5).

[0337] The solution FRET assay for YOP was also used to determine theIC₅₀ of ortho vanadate, a general protein phosphatase inhibitor. The YOPassay was performed as above, using 3 units of YOP and incubating theenzyme with the ZAP-GFP/peptide FRET mixture at 30° C. Dephosphorylationwas followed in real-time by measuring the increase in fluorescence at520 nm. Sodium orthovanadate was added to the reaction mix prior toenzyme addivity to give final concentrations of (0.03-30 μM). Resultsindicate an IC₅₀ value for vanadate of 0.25 μM and are shown in FIG. 6.

EXAMPLE6 Assay of Chk1 Kinase Using a Solution Phase FP Assay withFluorescein Labelled CDC25 Derived Peptide Substrate and 14-3-3ζ BindingPartner

[0338] This assay was performed to measure Chk1 activity by measuringthe fluorescence polarisation change that occurs as a result of the Chk1protein kinase-mediated binding of fluorescein labelled Chktide to14-3-3 protein.

[0339] Chk1 protein kinase modifies the activity of CDC25 phosphatasevia serine phosphorylation. Phosphorylation of CDC25 results in thebinding of different isoforms of the 14-3-3 protein and subsequentinhibition of CDC25 phosphatase activity. A peptide derived from CDC25(Chktide) and labelled with a chemical fluorophore binds to 14-3-3isoforms zeta and epsilon when phosphorylated by Chk1 kinase. Theactivity of Chk1 is monitored by following the fluorescence polarisationchange when fluorescein labelled Chktide binds to 14-3-3 protein.

[0340] Methods

[0341] Reagent Source

[0342] 14-3-3 ζ protein, Chk1 enzyme and Ckhtide peptide are fromUpstate Biotechnology.

[0343] Chktide , Chk1 substrate peptide sequence:

[0344] KKKVSRSGLYRSPS²¹⁶MPENLNRPR

[0345] Chktide Labelling with Fluorescein

[0346] Chktide was labelled by incubating the peptide for 2 hours at aconcentration of 0.185 mM in 100 mM NaHCO₃, pH 8.3, and 0.37 mMfluorescein-5EX (Molecular Probes) at room temperature. The labelledpeptide was then dialysed against 3 changes of 50 mM Tris HCL pH 7.4,150 mM NaCl (200 ml) for a total of 18 hours.

[0347] Phosphoryation of Chktide by Chk1 Protein Kinase.

[0348] A peptide substrate for Chk1 (Chktide) was labelled withfluorescein. The labelled peptides were then phosphorylated byincubating them at a concentration of 30 μM for 30 min at 30° C. in 20mM MOPS pH 7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mMsodium orthovanadate, 1 mM DTT, 0.1 mM ATP, and 62 mU Chk1. Nonphosphorylated control samples were prepared by incubation of thelabelled peptides under identical conditions in the absence of Chk1. Thepeptides were then incubated at a concentration of 5 μM in 20 mM MOPS pH7.2 at 30° C., in a total volume of 30 μl on a half area 96 well plate.The fluorescence polarisation of the samples was measured at 520 nm(excitation 485 nm) following a 5 min equilibration. 14-3-3 ζ (5 μM) wasadded to the peptide samples, and the fluorescence polarisation at 520nm (excitation 485 nm) was monitored over time as shown in FIG. 7.Inhibition of Chk1 activity by EDTA was measured by following the aboveprocedure and adding EDTA (10 mM or 20 mM) prior to enzyme addition, asshown in FIG. 8.

[0349] Activity of Chk1 was monitored in real time as follows.Fluorescein labelled Chktide was incubated at 30° C. in a total volumeof 30 μl (on a half area 96 well plate) in 20 mM MOPS pH 7.2, 10 mMMgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1mM DTT, and 0.1 mM ATP in the presence of 5 μM 14-3-3 ζ. The sampleswere allowed to equilibrate for 5 min, then Chk1 was added (or an equalvolume of H₂O for control samples) and the fluorescence polarisation at520 nm (excitation 485 nm) was monitored over time as shown in FIG. 9.Dependence of the increased fluorescence polarisation signal on Chk1activity and 14-3-3ζ binding was shown by the lack of binding when ATPor 14-3-3ζ protein was omitted from the real time enzyme reaction asshown in FIG. 10. Inhibition of Chk1 activity by EDTA was measured byfollowing the above procedure and adding EDTA (1 mM, 5 mM or 20 mM)prior to the addition of 21 mU of Chk1 to start the reaction, as shownin FIG. 11. The fluorescence polarisation for each sample was determinedat the end of the linear portion of the reaction.

[0350] Chk1 activity was also measured by monitoring the binding ofphosphorylated Chktide peptide to an alternate isoform of the 14-3-3protein, 14-3-3∈.

[0351] Production of 14-3-3 ∈

[0352] Under the control of the T7 promoter, the vector FS121 containsDNA encoding the 14-3-3∈ (Genbank accession number U54778) protein fusedin-frame to DNA encoding an amino terminal hexa-His tag. Freshtransformants of pFS121 in BRL(DE3) pLysS were used to inoculate 3 mlLB/ampicillin (100 μg/ml). The starter cultures was incubated overnightat 37° C. with shaking. From these starter cultures 1 ml was used toinoculate 400 ml Terrific Broth/ampicillin (100 μg/ml) in a 2L, baffledflask. Cultures were incubated at 37° C. at 200 rpm for approximately 4hr until the OD_(600nm) reached 0.5 Abs units. At this point cultureswere induced by adding IPTG to a concentration of 1 mM and furtherincubated at 37° C. for 4 hrs.

[0353] Bacteria were harvested by centrifugation at 3000 rpm for 20 min.The bacterial pellet was resuspended in 25 ml lysis buffer (50 mMPhosphate pH 7.0, 300 mM NaCl, 2% Proteinase inhibitor cocktail(Sigrna), 0.75 mg/ml Lysozyme). Lysis of the resuspended cells wasinitiated by gentle stirring for 30 min at room temperature. NonidetP-40 was added to a final concentration of 1% and lysis was continuedfor an additional 20 min at room temperature. The partially lysedmixture was subjected to 3 cycles of freeze thawing in liquid nitrogen.Finally the cells were sonicated on ice using a 10 mm probe at highpower. Sonication was performed on a pulse setting for a period of 4min. The crude lysate was centrifuged at 15,000 rpm for 30 min to removecell debris. Hexa-His tagged proteins were purified from the clearedlysate using TALON® resin (Clontech). Proteins were bound to the resinin a batchwise manner by gentle shaking at room temperature for 30 min.Non-His tagged proteins were removed by washing the resin at least twicewith a 10× bed volume of wash buffer (50 mM sodium phosphate pH 7.0, 300mM NaCl, 5 mM fluorescence-blank Imidazole ). The washed resin wasloaded into a 2 ml column and the bound proteins were released withelution buffer (50 mM sodium phosphate, pH 7.0, 300 mM NaCl, 150 mMfluorescence-blank Inidazole). Elution was normally achieved within 5ml. Purified proteins were stored at −80° C. after snap freezing inliquid nitrogen in the presence of 10% glycerol.

[0354] End Point Assay of Chk1 Kinase by 14-3-3∈ Binding toPhosphorylated Chktide

[0355] A peptide substrate for Chk1 (Chktide) was labelled withfluorescein. The labelled peptides were then phosphorylated byincubating them at a concentration of 30 μM for 30 min at 30° C. in 100μl of 20 mM MOPS pH 7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate, 5 mMEGTA, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mM ATP, and 62 mU Chk1.Non-phosphorylated control samples were prepared by incubation of thelabelled peptides under identical conditions in the absence of Chk1. Thepeptides were then incubated at a concentration of 7.5 μM in 20 mM MOPSpH 7.2 at 30° C., in a total volume of 30 μl on a half area 96 wellplate. The fluorescence polarisation of the samples was measured at 520nm (excitation 485 nm) following a 5 min equilibration. 14-3-3 ∈ (4 μl)was added to the peptide samples, and the fluorescence polarisation at520 nm (excitation 485 nm) was monitored over time as shown in FIG. 12.

EXAMPLE 7 Solution Phase FP Assay for the Detection of Phosphatase λActivity Using a Fluorescein Labelled CDC25 Derived Peptide Substrateand 14-3-3ζ Binding Partner

[0356] These assays were performed to demonstrate measurement ofphosphatase λ activity as detected by a change in fluorescencepolarisation due to decreased binding of Chktide to 14-3-3 ζ or 14-3-3∈. Binding is decreased as a result of dephosphorylation of Chktide byphosphatase λ.

[0357] Reagents were obtained and prepared as in example 6 above.

[0358] Fluorescein labelled Chktide was phosphorylated by incubation ata concentration of 30 μM for 30 min at 30° C. in 20 mM MOPS pH 7.2, 10mM MgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodiumorthovanadate, 1 mM DTT, 0.1 mM ATP, and 62 mU Chk1. Non-phosphorylatedcontrol samples were prepared by incubation of the labelled peptidesunder identical conditions in the absence of Chk1. The peptides werethen incubated at a concentration of 7.5 μM in 20 mM MOPS pH 7.2, 2 mMMnCl₂, 1 mM DTT at 30° C., in a total volume of 30 μl on a half area 96well plate. The fluorescence polarisation of the samples was measured at520 nm (excitation 485 nm) following a 5 min equilibration. 14-3-3 ζ (5μM) was added to the peptide samples, and the fluorescence polarisationat 520 nm (excitation 485 nm) was monitored over time as shown in FIG.13. Alternatively 14-3-3 ∈ (4 μl of purified protein prepared as inExample 6) was added to the peptide samples, and after the fluorescencepolarisation was stabilised, phosphatase λ(200U) was added. Thefluorescence polarisation at 520 nm (excitation 485 nm) was monitoredover time as shown in FIG. 14.

EXAMPLE 8 Simultaneous Assay of Two Serine Threonine Kinases, Chk1 andcAMP Dependent Protein Kinase (PKA) by FP

[0359] This assay was performed to simultaneously measure the activityof Chk1 and PKA by monitoring a change in fluorescence polarisation dueto association or dissociation of peptides and/or proteins capable ofassociating in a manner that is dependent upon their phosphorylationstate. Following phosphorylation by PKA, coiled coil dimers can nolonger form. Conversely, phosphorylation of Chktide by Chk1 results inbinding of Chktide to a 14-3-3 protein.

[0360] Serine threonine kinases PKA and Chk1 can be assayedsimultaneously using the natural binding partner reporters described inexample 5 for Chk1 and a peptide based binding partner assay for PKA(described in WO99/11774).

[0361] Methods

[0362] PKA Peptide sequences:

[0363] Peptide 1. ERE IKALERE IRRLRRA SQALERE IAQLERE

[0364] Peptide 2. LRQR IQCLRYR IRRLRRA SQALRQR IAQLKQR

[0365] PKA peptides form coiled-coil dimers when they arenon-phosphorylated. Each monomer has a PKA phosphorylation site.Following phosphorylation by PKA, the dimers can no longer form. Theassociation/disassociation can be measured using a fluorescencepolarisation assay, where PKA peptide 1 is labelled with coumarin, andpeptide 2 is labelled with biotin and bound to streptavidin. Thetumbling rate of coumarin labelled peptide 1 is higher after PKAphosphorylation and dissociation from the dimer/streptavidin complex. Atthe same time, the activity of Chk1 is measured by the decrease intumbling rate of phosphorylated fluorescein labelled Chktide substratebinding to 14-3-3∈ protein.

[0366] The labelled PKA peptides (both at a concentration of 2.5 μM)were incubated at 30° C. in a total volume of 50 μl (on a half area 96well plate) in 20 mM MOPS pH 7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate,5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, and 0.1 mM ATP, 7.5 μMChktide, 3 μL 14-3-3 ∈ and 0.06U streptavidin. The samples were allowedto equilibrate for 5 min, then CHK1 (62 mU) and PKA (0.5 pmoles) wereadded (or an equal volume of H₂O for control samples). The fluorescencepolarisation at 520 nm (excitation 485 nm) and at 450nm (excitation 340nm) was monitored over time as shown in FIG. 15.

EXAMPLE 9 FRET Assay of Src Using ZAP70-GFP Binding Partner andSynthetic Rhodamine Labelled TCRζ Substrate

[0367] These FRET-based assays were performed to detect Src activity bymeasuring the Src dependent formation of a complex between an SH2 domainof ZAP-70 labelled with a fluorophore and a peptide based on the TCRζchain labelled with a fluorophore.

[0368] A FRET partnership can be formed in a phosphorylation dependentmanner between the SH2 domain of ZAP-70 and a peptide based on the TCRζchain, providing both binding partners are labelled with suitablefluorophores. Src is used to phosphorylate the TCRζ chain derivedsubstrate.

[0369] Methods:

[0370] TCR Pepide Sequence

[0371] Peptide 1. Phosphorylated TCRζ chain:

[0372] RCKFSRSAEPPAYQQGQNQLY_((p))NELNLGRREEY_((p))DVLD

[0373] Peptide 2. Unphosphorylated TCRζ chain:

[0374] RCKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLD

[0375] Peptide Labelling

[0376] Peptides 1 and 2 were labelled with rhodamine under mildconditions using thiol directed chemistry. 230 μM peptide was labelledin 20 mM TES pH 7 in the presence of a three-fold excess ofrhodamine-6-maleimide (Molecular Probes). Dialysis was utilised toremove excess dye from the peptide. Labelling was verified by MALDI-TOFMS.

[0377] ZAP-GFP Cloning and Purification

[0378] ZAP-GFP was cloned and purified as described in Example 5.

[0379] Phosphorylation of Peptide 2

[0380] Peptide 2 labelled with rhodamine was phosphorylated using 3units of Src kinase (Upstate Biotechnology) in a 40 μl reactioncontaining 115 μM peptide, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10 mM MgCl₂,10 mM β-mercaptoethanol, 0.1 mg/ml BSA and 0.015% (v/v) Brij 35, over aperiod of 2 hours at 37° C. Non-phosphorylated control samples wereprepared under identical conditions in the absence of the kinase.

[0381] Formation of the FRET Partnership

[0382] ZAP-GFP was diluted to 0.5 μM in assay buffer (50 mM Tris pH 7.2,10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35). 98 μl of thissolution was used per assay. Initial readings of the fluorescence of theZAP-GFP construct were made using 485 nm excitation wavelength and 520nm emission wavelength. Rhodamine labelled peptide (2 μl of abovephosphorylation reactions) was added to the ZAP-GFP solution andformation of FRET was followed in real-time by measuring the decrease influorescence emission of ZAP-GFP at 520 nm as shown in FIG. 16.

EXAMPLE 10 Solution Phase FRET Assay of Src Kinase Activity Using aNatural Binding Partner SHP-2 Labelled with a Fluorescent Protein

[0383] These FRET-based assays are performed to measure Src kinaseactivity as determined by the Src kinase dependent formation of acomplex between the SH2 domain of SHP2 labelled with a fluorophore andan synthetic peptide based on SHPS-1, also labelled with a fluorophore.

[0384] Interaction between the tandem SH2 domain of SHP2 and a syntheticpeptide based on SHPS-1 is mediated by the state of phosphorylation ofthe peptide. A FRET partnership can be established betweenphosphorylated peptide and the SH2 domain providing both moieties arelabelled with suitable fluorophores which are brought into closeproximity upon interaction of binding partners.

[0385] Methods:

[0386] SHP2 Peptide Sequence and Fluorescent Labelling

[0387] SHPS-1 peptide sequence.

[0388] BiotinKQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSC

[0389] The SHPS-1 was labelled using thiol directed chemistry. 200 μMpeptide was reacted with 600 μM rhodamine-6-maleimide (Molecular Probes)in 20 mM TES pH 7 over a period of at least two hours at roomtemperature. Excess label was removed using dialysis and the labellingwas verified by MALDI-TOF MS.

[0390] SHP2-GFP Cloning and Purification

[0391] Primers were based on the published SHP-2 DNA sequence (Genbankaccession number L03535. The SH2 domain (amino acids 1-225) of SHP-2 wascloned by PCR using the following oligo-nucleotides: 5′ primer-GGGGATCCTCTAGAATGACATCGCGGAGATGGTTTCACCC 3′ primer-GGGGAATTCTTTCAGCAGCATTTATACGAGTCG

[0392] The resultant PCR fragment was digested with XbaI and EcoRI, gelpurified and ligated into pET28a (Novagen) to generate vector pFS 114.The validity of the construct was confirmed by sequence analysis. DNAencoding GFP in the vector pFS46 was isolated by digestion with therestriction enzymes EcoRI and XhoI and the resultant 860 bp band was gelpurified and ligated into pFS114 to generate a bacterial expressionvector for production of the fusion protein SHP2-GFP (pFS115).

[0393] The hexa-His tagged SHP2-GFP fusion protein was expressed andpurified using TALON® resin according to standard procedures.

[0394] Phosphorylation of Peptide SHPS-1 Peptide

[0395] SHPS-1 peptide labelled with rhodamine was phosphorylated using 3units of Src kinase (Upstate Biotechnology) in a 40 μl reactioncontaining 100 μM peptide, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10 mM MgCl₂,10 mM β-mercaptoethanol, 0.1 mg/ml BSA and 0.015% (v/v) Brij 35, over aperiod of 2 hours at 37° C. Non-phosphorylated control samples wereprepared under identical conditions in the absence of the kinase.

[0396] Formation of the FRET Partnership

[0397] SHP2-GFP was diluted to 0.5 μM in assay buffer (50 mM Tris HCL pH7.2, 10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35). 98 μl ofthis solution was used per assay. Initial readings of the fluorescenceof the SHP2-GFP construct were made using 485 nm excitation wavelengthand 520 nm emission wavelength. Rhodamine labelled peptide (2 μl of theabove phosphorylation reactions) was added to the SHP2-GFP solution andformation of FRET was monitored in real-time by measuring the decreasein fluorescence emission of SHP2-GFP at 520 nm as shown in FIG. 17.

[0398] Disruption of the FRET Partnership

[0399] The FRET partnership described in the above section was disruptedeasily using the tyrosine phosphatase enzyme, YOP (UpstateBiotechnology). FRET partnership was formed as in the above example,using 4 μl of phosphorylated or control non-phosphorylated SHPS-1peptide. Addition of 3 units of YOP to the FRET partnership resulted indisruption of the FRET partnership as the phosphotyrosines required forthe formation of the partnership were removed (shown in FIG. 18).

[0400] Inhibition by Staurosporine

[0401] Phosphorylation of the peptide was prevented by inhibiting theenzyme, Src, with the potent kinase inhibitor staurosporine.Phosphorylation of the rhodamine labelled SHPS-1 peptide was performedas described above in the presence or absence of 10 μM staurosporineadded to the reaction prior to the addition of the Src enzyme. The SH2domain of SHP2 and the SHPS-1 peptide failed to form a FRET partnershipin the presence of the inhibitor, as shown in FIG. 19.

[0402] Assay of Src Phosphorylation of SHPS-1 Peptide in Real-Time

[0403] Phosphorylation of the rhodamine labelled SHPS-1 peptide andformation of the FRET partnership with SHP2-GFP was followed inreal-time by measuring the decrease in fluorescence emission at 520 nm.Reactions containing 0.5 μM SHP2-GFP, 50 mM Tris-HCl pH 7.2, 1 mM ATP,10 mM MgCl₂, 10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35 and40 μM peptide were set up in a black microtitre plate. An initialequilibrium measurement was made before adding 6 units of Src kinase, orbuffer to control wells. The decrease in fluorescence emission at 520 nmwas followed in real-time at 37° C. as shown in FIG. 20.

EXAMPLE 11 In Vivo Measurement of Kinase or Phosphatase Activity

[0404] The enzymatic activity of a kinase or phosphatase enzyme ismeasured in an in vivo assay performed as follows.

[0405] In vivo assays are carried out by transfecting cells with a firstexpression construct encoding a fusion protein comprising a polypeptidecomprising a natural binding domain and further comprising a site forphosphorylation fused in frame to a fluorescent protein and a secondexpression construct comprising a polypeptide comprising a bindingpartner for the natural binding domain fused in frame to a secondfluorescent protein. Alternatively, cells are transfected with a tandemconstruct encoding a fusion protein comprising a natural binding domainthat includes a site for phosphorylation and a binding partner for thenatural binding domain, and two different fluorescent proteins. For allexperiments, binding of a natural binding domain to its binding partneris dependent on phosphorylation or dephosphorylation.

[0406] Plasmids encoding the autofluorescent proteins (AFPs) red shiftedgreen fluorescent protein (rsGFP) and blue fluorescent protein (BFP) arepurchased from Quantum Biotechnologies, Inc. BFP is a mutated version ofthe 28 kDa rsGFP. BFP has an excitation peak of 387 nm and an emissionpeak of 450 nm. GFP has an excitation peak of 473 nm and an emissionpeak of 509 nm.

[0407] Constructs

[0408] DNA primers are designed encoding a first polypeptide comprisinga natural binding and a phosphorylation site domain or a secondpolypeptide comprising a binding partner for the first polypeptide, astop codon and unique restriction sites (e.g. BamHI and EcoRI) at eachend to facilitate cloning. Codon usage is selected in order to allowboth mammalian and bacterial expression. Alternatively, DNA primers aredesigned to encode a polypeptide comprising both a natural bindingdomain that includes a phosphorylation site and a binding partner forthe natural binding domain.

[0409] Experiments are performed using the following pair ofpolypeptides:

[0410] 1. v-SRC SH2 domain (amino acids 148-246; Waksman et al., supra;OWL database accession no. M33292 and hamster polyomavirus middle Tantigen (Ag) (321-331, EPQYEEIPIYL), Waksman et al., supra; OWL databaseaccession no. P03079,

[0411] 2. Phosphotyrosine binding domain (PTB) of IRS-1 (amino acids157-267) Zhou et al., supra; OWL accession no. P35568 and interleukin 4receptor (I1-4R) (amino acids 489-499, LVIAGNPAYRS; Zhou et al., supra;OWL database accession no. P24394, and

[0412] 3. The PTB domain of the proto-oncogene product Cb1 (the Cb1N-terminal binding domain) (amino acids 1-357); Lupher et al., supra;OWL accession no. P22681 and a peptide derived from the Zap-70 tyrosinekinase (amino acids 284-299, NH₃-IDTLNSDGYtpepARI-COOH); Lupher et al.,supra;.OWL accession no. P43403.

[0413] 4. SH2 domain of ZAP70 (residues 1-259), GenBank accession No.L05148. Tandem phosphorylation motif of TCRseta chain (residues 52-163)GenBank accession No. J04132.

[0414] Experiments are also performed using a construct encoding apolypeptide comprising a natural binding domain including a site forphosphorylation and further comprising a natural binding partner for thenatural binding domain.

[0415] These experiments are performed using the polypeptide c-SRC(residues 86-536); Xu et al., supra; GenBank Accession No. K03218.

[0416] AFP-Polypetide Construction

[0417] The purified DNA fragment, isolated by PCR is digested with theappropriate enzymes that cleave at the unique restriction sites locatedat each end and purified as above prior to ligation into the mammalianexpression vectors pQBI25-fc1 and pQBI50-fc1.

[0418] The v-SRC-SH2 domain and the polyomavirus middle T-antigenpeptide are cloned such that the AFP is placed at the N-termini. TheAFPs can be fused either to the N or C-termini of the PTB domain ofIRS-1 and the IL-4R peptide.

[0419] In the case of the tandem construct expressing the c-SRCpolypeptide which includes both a natural binding domain, including asite of phosphorylation and a natural binding partner for the naturalbinding domain, chemical modification by the kinase or phosphataseenzyme being assayed is monitored by measuring a change in anintramolecular reaction. A nucleic acid encoding a natural bindingdomain including a site of phosphorylation and its binding partner to beexpressed as part of a single-polypeptide, additionally encodes, at aminimum, a donor AFP fused to the natural binding domain and an acceptorAFP fused to its binding partner, a linker that couples the two AFPs andis of sufficient length and flexibility to allow for folding of thepolypeptide and pairing of the natural binding domain, sequence orpolypeptide with the binding partner, and gene regulatory sequencesoperatively linked to the fusion coding sequence.

[0420] To prepare a construct encoding a polypeptide comprising anatural binding domain and further comprising a natural binding partnerfor the natural binding domain and two different AFP proteins, thepurified DNA (prepared as above) is ligated into a vector encoding anAFP. A fragment encoding the polypeptide plus an AFP protein is excisedfrom the vector and ligated into a second vector encoding a linker and asecond AFP. Alternatively, the purified DNA (prepare as above) isligated into a vector encoding an AFP. A DNA fragment encoding a linkerand a second AFP is ligated into the above construct (by digestion withappropriate restriction enzymes) resulting in a construct encoding apolypeptide comprising a natural binding domain, a natural bindingpartner for the natural binding domain and two AFPs separated by alinker.

[0421] FRFT in Mammalian Cells

[0422] Experiments are performed using pairs of vectors expressing thefollowing proteins: a polypeptide comprising a natural binding domainincluding a phosphorylation site and an AFP and a polypeptide comprisinga natural binding partner of the natural binding domain and a secondAFP.

[0423] Vectors capable of expressing these proteins are transfected intoCOS-7 cells (a well-established cell-line derived from monkey kidneycells) individually and in combination. Transfections are performedusing Lipofectamine 2000 (GibcoBRL) and the transfected cells areincubated at 37° C. for 48 hr (to allow the expressed proteins toaccumulate to a detectable level) in the presence or absence of a kinaseactivator, a candidate modulator of kinase activity or both a kinaseactivator and a candidate modulator of kinase activity. Alternatively,the transfected cells are incubated in the presence or absence of aphosphatase activator, a candidate modulator of phosphatase activity orboth a phosphatase activator and a candidate modulator of phosphataseactivity.

[0424] Additional experiments are performed (using the transfectionprotocol described above) using a tandem vector expressing a polypeptidecomprising a natural binding domain including a phosphorylation site, anatural binding partner of the natural binding domain and two differentAFPs separated by an appropriate linker. As above, transfected cells areincubated for 48 hrs in the presence or absence of a kinase, a candidatemodulator of kinase activity, both a kinase and a candidate modulator ofkinase activity, a phosphatase, a candidate modulator of phosphataseactivity or both a phosphatase and a candidate modulator of phosphataseactivity.

[0425] Following the 48 hr incubation period the amount of FRET isdetermined by analysis in a BMG Galaxy fluorescent plate reader usingthe following regime: excitation at 370 nm (excitation for BFP) andemission at 520 nm (emission for GFP). USE

[0426] The invention is useful in monitoring the activity of a proteinkinase or phosphatase, whether the protein is isolated,partially-purified, present in a crude preparation or present in aliving cell. The invention is further useful in assaying a cell or cellextract for the presence- or level of activity of a protein kinase orphosphatase. The invention is additionally useful in assaying theactivity of naturally-occurring (mutant) or non-natural (engineered)isoforms of known protein kinases and/or phosphatases or, instead, thatof novel (natural or non-natural) enzymes. The invention is of use inassaying the efficacy of candidate modulators of the activity of aprotein kinase or phosphatase in inhibiting or enhancing the activity ofthat enzyme; moreover, is useful to screen potential therapeutic drugsfor activity against cloned and/or purified enzymes that may haveimportant clinical pathogenicities when mutated. The invention isfurther of use in the screening of a candidate bioactive agent (e.g.,drugs) for side effects, whereby the ability of such an agent tomodulate the activity of a protein kinase or phosphatase may beindicative a propensity toward provoking unintended side-effects to atherapeutic or other regimen in which that agent might be employed.

OTHER EMBODIMENTS

[0427] Other embodiments will be evident to those of skill in the art.It should be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

What is claimed is:
 1. An isolated natural binding domain and a bindingpartner therefor, wherein said isolated natural binding domain includesa site for post-translational phosphorylation and binds said bindingpartner in a manner dependent upon phosphorylation or dephosphorylationof said site.
 2. The isolated natural binding domain and binding partnertherefor of claim 1, wherein said phosphorylation or dephosphorylationis performed by an enzyme which is a kinase or a phosphatase,respectively.
 3. The isolated natural binding domain and binding partnertherefor of claim 1, wherein phosphorylation of said site preventsbinding of said isolated natural binding domain to said binding partner.4. The isolated natural binding domain and binding partner therefor ofclaim 1, wherein phosphorylation of said site promotes binding of saidisolated natural binding domain to said binding partner.
 5. The isolatednatural binding domain and binding partner therefor of claim 1, whereindephosphorylation of said site prevents binding of said isolated naturalbinding domain to said binding partner.
 6. The isolated natural bindingdomain and binding partner therefor of claim 1, whereindephosphorylation of said site promotes binding of said isolated naturalbinding domain to said binding partner.
 7. The isolated natural bindingdomain and binding partner therefor of claim 1, wherein at least one ofsaid isolated natural binding domain and said binding partner comprisesa detectable label.
 8. The isolated natural binding domain and bindingpartner therefor of claim 7, wherein said detectable label emits light.9. The isolated natural binding domain and binding partner therefor ofclaim 8, wherein said light is fluorescent.
 10. The isolated naturalbinding domain and binding partner therefor of claim 9, wherein one ofsaid isolated natural binding domain and said binding partner comprisesa quencher for said detectable label.
 11. A kit comprising an isolatednatural binding domain and a binding partner therefor, wherein saidisolated natural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner dependentupon phosphorylation of said site, and packaging material therefor. 12.The kit of claim 11, wherein said kit further comprises a buffer whichpermits phosphorylation-dependent binding of said isolated naturalbinding domain and said binding partner.
 13. The kit of claim 12,wherein said buffer permits phosphorylation or dephosphorylation of saidsite by a kinase or a phosphatase, respectively.
 14. The kit of claim11, wherein said kit further comprises one or both of a kinase and aphosphatase.
 15. The kit of claim 14, wherein said kit further comprisesa substrate for said phosphatase or kinase, said substrate being MgATP.16. The kit of claim 14, wherein said kit further comprises a cofactorfor one or both of said kinase and phosphatase.
 17. The kit of claim 11,wherein at least one of said isolated natural binding domain and saidbinding partner comprises a detectable label.
 18. The kit of claim 17,wherein said detectable label emits light.
 19. The kit of claim 18,wherein said light is fluorescent.
 20. A method for monitoring activityof an enzyme comprising performing a detection step to detect binding ofan isolated natural binding domain and a binding partner therefor as aresult of contacting one or both of said isolated natural binding domainand said binding partner with said enzyme, wherein said isolated naturalbinding domain includes a site for post-translational phosphorylationand binds said binding partner in a manner dependent uponphosphorylation of said site and wherein detection of binding of saidisolated natural binding domain and said binding partner as a result ofsaid contacting is indicative of enzyme activity.
 21. A method formonitoring activity of an enzyme comprising performing a detection stepto detect dissociation of an isolated natural binding domain from abinding partner therefor as a result of contacting one or both of saidisolated natural binding domain and said binding partner with saidenzyme, wherein said isolated natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner dependent upon phosphorylation of said site and wherein detectionof dissociation of said isolated natural binding domain from saidbinding partner as a result of said contacting is indicative of enzymeactivity.
 22. The method of claim 20 or 21, wherein at least one of saidisolated natural binding domain and said binding partner is labeled witha detectable label.
 23. The method of claim 22, wherein said label emitslight.
 24. The method of claim 23, wherein said light is fluorescent.25. The method of claim 22 wherein said detection step is to detect achange in signal emission by said detectable label.
 26. The methodaccording to claim 25, wherein said method further comprises excitingsaid detectable label and monitoring fluorescence emission.
 27. Themethod according to claim 25, wherein said method further comprises thestep, prior to or after said detection step, of contacting said isolatednatural binding domain and said binding partner with an agent whichmodulates the activity of said enzyme.
 28. A method for monitoring theactivity of a modulator of the activity of an enzyme comprising: a)mixing an isolated natural binding domain, a binding partner of saidisolated natural binding domain, said enzyme, and a candidate modulatorwhich binds to said isolated natural binding domain, wherein saidisolated natural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner dependentupon phosphorylation of said site, and wherein the combination of saidisolated natural binding domain and said binding partner comprisesdetection means for monitoring association or dissociation of saidisolated natural binding domain and said binding partner, and whereindetection of binding of said isolated natural binding domain and saidbinding partner as a result of said mixing is indicative of enzymeactivity, and wherein said phosphorylation of said site occurs prior tosaid mixing step; and b) monitoring association or dissociation of saidisolated natural binding domain and said binding partner, saidassociation or dissociation being indicative of modulation by saidcandidate modulator of said activity, wherein said modulator reducesbinding of said isolated natural binding domain and said binding partnerand wherein a reduction in binding is detected by said detection means.29. A method for monitoring the activity of a modulator of the activityof an enzyme comprising: a) mixing an isolated natural binding domain, abinding partner of said isolated natural binding domain, said enzyme,and a candidate modulator which binds to said isolated natural bindingdomain, wherein said isolated natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner dependent upon phosphorylation of said site, and wherein thecombination of said isolated natural binding domain and said bindingpartner comprises detection means for monitoring association ordissociation said isolated natural binding domain and said bindingpartner, and wherein detection of binding of said isolated naturalbinding domain and said binding partner as a result of said mixing isindicative of enzyme activity, and wherein said phosphorylation of saidsite occurs during said mixing step; and b) monitoring association ordissociation of said isolated natural binding domain and said bindingpartner, said association or dissociation being indicative of modulationby said candidate modulator of said activity, wherein said modulatorreduces binding of said isolated natural binding domain and said bindingpartner and wherein a reduction in binding is detected by said detectionmeans.
 30. A method for monitoring the activity of a modulator of theactivity of an enzyme comprising: a) mixing an isolated natural bindingdomain, a binding partner of said isolated natural binding domain, saidenzyme, and a candidate modulator which binds to said binding partner,wherein said isolated natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner dependent upon phosphorylation of said site, and wherein thecombination of said isolated natural binding domain and said bindingpartner comprises detection means for monitoring association ordissociation of said isolated natural binding domain and said bindingpartner, and wherein detection of binding of said isolated naturalbinding domain and said binding partner as a result of said mixing isindicative of enzyme activity, and wherein said phosphorylation of saidsite occurs prior to said mixing step; and b) monitoring association ordissociation of said isolated natural binding domain and said bindingpartner, said association or dissociation being indicative of modulationby said candidate modulator of said activity, wherein said modulatorreduces binding of said isolated natural binding domain and said bindingpartner and wherein a reduction in binding is detected by said detectionmeans.
 31. A method for monitoring the activity of a modulator of theactivity of an enzyme comprising: a) mixing an isolated natural bindingdomain, a binding partner of said isolated natural binding domain, saidenzyme, and a candidate modulator which binds to said binding partner,wherein said isolated natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner dependent upon phosphorylation of said site, and wherein thecombination of said isolated natural binding domain and said bindingpartner comprises detection means for monitoring association ordissociation of said isolated natural binding domain and said bindingpartner, and wherein detection of binding of said isolated naturalbinding domain and said binding partner as a result of said mixing isindicative of enzyme activity, and wherein said phosphorylation of saidsite occurs during said mixing step; and b) monitoring association ordissociation of said isolated natural binding domain and said bindingpartner, said association or dissociation being indicative of modulationby said candidate modulator of said activity, wherein said modulatorreduces binding of said isolated natural binding domain and said bindingpartner and wherein a reduction in binding is detected by said detectionmeans.
 32. The method of claim 28, 29, 30 or 31, wherein at least one ofsaid isolated natural binding domain and said binding partner is labeledwith a detectable label.
 33. The method of claim 32, wherein said labelemits light.
 34. The method of claim 33, wherein said light isfluorescent.
 35. The method of claim 34, wherein said detection step isto detect a change in signal emission by said detectable label.
 36. Themethod of claim 35, wherein said method further comprises exciting saiddetectable label and monitoring fluorescence emission.
 37. The method ofclaim 35, wherein said method further comprises the step, prior to orafter said detection step, of contacting said isolated natural bindingdomain and said binding partner with an agent which modulates theactivity of said enzyme.
 38. A method of screening for a candidatemodulator of enzymatic activity of a kinase or a phosphatase, the methodcomprising a) mixing an isolated natural binding domain, a bindingpartner therefor and an enzyme with a candidate modulator of said kinaseor phosphatase which binds said isolated natural binding domain, whereinsaid natural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner that isdependent upon phosphorylation or dephosphorylation of said site by saidkinase or phosphatase and wherein the combination of said isolatednatural binding domain and said binding partner comprises a detectionmeans for monitoring association or dissociation between said isolatednatural binding domain and said binding partner, and wherein saidphosphorylation or dephosphorylation occurs prior to said mixing, and b)monitoring the association or dissociation of said isolated naturalbinding domain to said binding partner, wherein association ordissociation of said isolated natural binding domain and said bindingpartner as a result of said contacting is indicative of modulation ofenzymatic activity by said candidate modulator of said kinase orphosphatase.
 39. A method of screening for a candidate modulator ofenzymatic activity of a kinase or a phosphatase, the method comprisinga) mixing an isolated natural binding domain, a binding partner thereforand an enzyme with a candidate modulator of said kinase or phosphatasewhich binds to said isolated natural binding domain, wherein saidnatural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner that isdependent upon phosphorylation or dephosphorylation of said site by saidkinase or phosphatase and wherein the combination of said isolatednatural binding domain and said binding partner comprises a detectionmeans for monitoring association or dissociation between said isolatednatural binding domain and said binding partner, and wherein saidphosphorylation or dephosphorylation occurs during said mixing, and b)monitoring the association or dissociation of said isolated naturalbinding domain to said binding partner, wherein association ordissociation of said isolated natural binding domain and said bindingpartner as a result of said contacting is indicative of modulation ofenzymatic activity by said candidate modulator of said kinase orphosphatase.
 40. A method of screening for a candidate modulator ofenzymatic activity of a kinase or a phosphatase, the method comprisinga) mixing an isolated natural binding domain, a binding partner thereforand an enzyme with a candidate modulator of said kinase or phosphatasewhich binds said binding partner, wherein said natural binding domainincludes a site for post-translational phosphorylation and binds saidbinding partner in a manner that is dependent upon phosphorylation ordephosphorylation of said site by said kinase or phosphatase and whereinthe combination of said isolated natural binding domain and said bindingpartner comprises a detection means for monitoring association ordissociation between said isolated natural binding domain and saidbinding partner, and wherein said phosphorylation or dephosphorylationoccurs prior to mixing, and b) monitoring the binding of said isolatednatural binding domain to said binding partner, wherein binding ordissociation of said isolated natural binding domain and said bindingpartner as a result of said contacting is indicative of modulation ofenzymatic activity by said candidate modulator of said kinase orphosphatase.
 41. A method of screening for a candidate modulator ofenzymatic activity of a kinase or a phosphatase, the method comprisinga) mixing an isolated natural binding domain, a binding partner thereforand an enzyme with a candidate modulator of said kinase or phosphatasewhich binds said binding partner, wherein said natural binding domainincludes a site for post-translational phosphorylation and binds saidbinding partner in a manner that is dependent upon phosphorylation ordephosphorylation of said site by said kinase or phosphatase and whereinthe combination of said isolated natural binding domain and said bindingpartner comprises a detection means for monitoring association ordissociation between said isolated natural binding domain and saidbinding partner, and wherein said phosphorylation or dephosphorylationoccurs during said mixing, and b) monitoring the association ordissociation of said isolated natural binding domain to said bindingpartner, wherein association or dissociation of said isolated naturalbinding domain and said binding partner as a result of said contactingis indicative of modulation of enzymatic activity by said candidatemodulator of said kinase or phosphatase.
 42. The method of claim 38, 39,40 or 41, wherein said detectable label emits light.
 43. The method ofclaim 42, wherein said light is fluorescent.
 44. The method of claim 43,wherein said monitoring comprises measuring a change in energy transferbetween a detectable label present on said isolated natural bindingdomain and a detectable label present on said binding partner.
 45. Amethod of screening for a candidate modulator of enzymatic activity of akinase or a phosphatase, the method comprising a) mixing an assay systemcomprising an isolated natural binding domain and a binding partner forsaid isolated natural binding partner with a candidate modulator ofenzymatic activity of a said kinase or phosphatase which binds to saidisolated natural binding domain, and b) monitoring association ordissociation of an isolated natural binding domain and a binding partnertherefor in said assay system, wherein said isolated natural bindingdomain includes a site for post-translational phosphorylation and bindssaid binding partner in a manner that is dependent upon phosphorylationor dephosphorylation of said site by a said kinase or phosphatase insaid assay system, wherein the combination of said isolated naturalbinding domain and said binding partner comprises a detection means formonitoring association or dissociation between said isolated naturalbinding domain and said binding partner and wherein said phosphorylationor dephosphorylation occurs prior to said mixing, and whereinassociation or dissociation of said isolated natural binding domain andsaid binding partner as a result of said contacting is indicative ofmodulation of enzymatic activity by said candidate modulator of a saidkinase or phosphatase.
 46. A method of screening for a candidatemodulator of enzymatic activity of a kinase or a phosphatase, the methodcomprising a) mixing an assay system comprising an isolated naturalbinding domain and a binding partner for said isolated natural bindingdomain with a candidate modulator of enzymatic activity of a said kinaseor phosphatase which binds to said isolated natural binding domain, andb) monitoring association or dissociation of an isolated natural bindingdomain and a binding partner therefor in said assay system, wherein saidisolated natural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner that isdependent upon phosphorylation or dephosphorylation of said site by asaid kinase or phosphatase in said assay system, wherein the combinationof said isolated natural binding domain and said binding partnercomprises a detection means for monitoring association or dissociationbetween said isolated natural binding domain and said binding partnerand wherein said phosphorylation or dephosphorylation occurs during saidmixing, and wherein association or dissociation of said isolated naturalbinding domain and said binding partner as a result of said contactingis indicative of modulation of enzymatic activity by said candidatemodulator of a said kinase or phosphatase.
 47. A method of screening fora candidate modulator of enzymatic activity of a kinase or aphosphatase, the method comprising a) mixing an assay system comprisingan isolated natural binding domain and a binding partner with acandidate modulator of enzymatic activity of a said kinase orphosphatase which binds to said binding partner, and b) monitoringassociation or dissociation of an isolated natural binding domain and abinding partner therefor in said assay system, wherein said isolatednatural binding domain includes a site for post-translationalphosphorylation and binds said binding partner in a manner that isdependent upon phosphorylation or dephosphorylation of said site by asaid kinase or phosphatase in said assay system, wherein the combinationof said isolated natural binding domain and said binding partnercomprises a detection means for monitoring association or dissociationbetween said isolated natural binding domain and said binding partnerand wherein said phosphorylation or dephosphorylation occurs prior tosaid mixing, and wherein association or dissociation of said isolatednatural binding domain and said binding partner as a result of saidcontacting is indicative of modulation of enzymatic activity by saidcandidate modulator of a said kinase or phosphatase.
 48. A method ofscreening for a candidate modulator of enzymatic activity of a kinase ora phosphatase, the method comprising a) mixing an assay systemcomprising an isolated natural binding domain and a binding partner witha candidate modulator of enzymatic activity of a said kinase orphosphatase which binds to said binding partner, and b) monitoringbinding of an isolated natural binding domain and a binding partnertherefor in said assay system, wherein said isolated natural bindingdomain includes a site for post-translational phosphorylation and bindssaid binding partner in a manner that is dependent upon phosphorylationor dephosphorylation of said site by a said kinase or phosphatase insaid assay system, wherein the combination of said isolated naturalbinding domain and said binding partner comprises a detection means formonitoring association or dissociation between said isolated naturalbinding domain and said binding partner and wherein said phosphorylationor dephosphorylation occurs during said mixing, and wherein associationor dissociation of said isolated natural binding domain and said bindingpartner as a result of said contacting is indicative of modulation ofenzymatic activity by said candidate modulator of a said kinase orphosphatase.
 49. The method of claim 28, 29, 30, 31, 38, 39, 40, 41, 45,46, 47, or 48, wherein said method comprises real-time observation ofassociation or dissociation of a said isolated natural binding domainand its binding partner.